Methods for treatment or prevention of damage resulting from radiation, trauma or shock

ABSTRACT

Described herein are methods of treating a subject that has been or will be exposed to radiation, trauma or shock, the method comprising identifying a subject that has been or will be exposed to radiation, and treating the subject with a compound that treats, reduces the severity or delays the onset of sepsis or reduces the likelihood of mortality in a subject upon administration of a therapeutically effective amount the compound to the subject.

CLAIMS OF PRIORITY

This application claims priority to U.S. Ser. No. 61/703,703, filed Sep.20, 2012 and U.S. Ser. No. 61/737,576, filed Dec. 14, 2012, the entirecontents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to the use of polyglucosamines to treat or preventdamage resulting from radiation, trauma or shock.

BACKGROUND

Environmental or physical stresses and stimuli, for example, fromradiation, trauma or shock, can result in biological damage or otherwisetrigger a series of intricate biological events that can increase themorbidity and mortality rate in a subject.

SUMMARY OF THE INVENTION

Methods of treating a subject that has been or will be exposed toradiation, trauma or shock are described herein. Exemplary methodsdescribed herein include, for example, methods of treating a subjectthat has been exposed to radiation, trauma or shock with a compound thatreduces sepsis or mortality; methods of reducing the severity of sepsisor a symptom thereof or decreasing the likelihood of mortality of asubject that has been or will be exposed to radiation, trauma or shock.

In an aspect, the invention features a method of treating a subject, themethod comprising identifying a subject that has been exposed toradiation, trauma or shock, and treating the identified subject byadministering to the identified subject a therapeutically effectiveamount of a compound to the subject, wherein the compound is a compoundof Formula (I):

wherein n is an integer between 20 and 6000; and each R¹ isindependently selected for each occurrence from hydrogen, acetyl,

wherein at least 25% of R¹ substituents are H, at least 1% of R¹substituents are acetyl, and atleast 2% of R¹ substituents are

wherein upon administration of the compound, the compound treats,reduces the severity or delays the onset of sepsis or reduces thelikelihood of mortality in the subject, thereby treating the subject.

In some embodiments, the subject has a bacterial infection, chemicaldamage or radiation damage, e.g., resulting in leaky gut and/or damageto the GI tract. In some embodiments, the infection or damage results inleaky gut.

In some embodiments, the method reduces the severity of sepsis or asymptom thereof or decreases the likelihood of mortality from theradiation, trauma or shock relative to a subject not administered withthe compound.

In some embodiments, the subject has sepsis or a symptom of sepsisresulting from radiation, trauma or shock. In some embodiments, thesepsis is caused by leaky gut (e.g., mucosal lesions). In someembodiments, the subject is at risk of developing sepsis as a result ofexposure to radiation, trauma or shock.

In some embodiments, the radiation, trauma or shock results in reducedintegrity of the GI tract of the subject or leaky gut (e.g., mucosallesions in the GI tract) in a subject.

In some embodiments, the trauma or shock is a bacterial, viral, orfungal infection resulting in GI damage. In some embodiments, thebacterial infection is from one of the following bacteria: Salmonellaenterica serovar Typhimurium, Shigella flexneri, E. coli and P.aeruginosa.

In some embodiments, the subject has been exposed to radiation in anamount sufficient to produce 30% to 80% lethality, e.g., at 30% to 80%lethal dose, at LD30 to LD80.

In some embodiments, the method reduces inflammation in the subject fromthe radiation, trauma or shock. In some embodiments, the methodmitigates the inflammatory response in the GI tract. In someembodiments, the method mitigates the inflammatory response and reducesmortality due to bacterial infection, bacterial translocation orchemical damage or radiation damage in the GI tract of a subjectrelative to a subject not administered with the compound.

In some embodiments, the method protects epithelial cells from bacterialinvasion. In some embodiments, the method reduces translocation ofbacteria across the GI tract, e.g., by up to 80%. In some embodiments,the compound acts through mucoadhesive substantivity (e.g., adhesion,affinity). In some embodiments, the method reduces crypt degeneration.In some embodiments, the method promotes the health of villousepithelium (e.g. reduces loss or blunting of villi).

In some embodiments, the method reduces mortality after exposure of theGI tract of a subject to ionizing radiation relative to a subject notadministered with the compound. In some embodiments, the radiation isfrom a dirty bomb, accidental nuclear incident or therapeutic radiationnot related to the treatment of cancer. In some embodiments, theradiation is targeted to therapeutic treatment requiring destruction ofthe immune system, e.g., bone marrow transplant therapy or otherelective exposure to radiation.

In some embodiments, the trauma results from exposure to a toxicchemical or poison. In some embodiments, the toxic chemical or poison isingested.

In some embodiments, the trauma results from multi-organ failure fromphysical damage and trauma to the body, burns, blast injury, systemicinfection, blood loss (hypotension), and traumatic brain injury.

In some embodiments, the shock is physical shock not resulting in awound. In some embodiments, the shock results from excessive stimuli,e.g. pathogens, commensals, injury, heat, autoantigens, tumors, necroticcells. In some embodiments, the trauma results from a commensal or apathogen translocating to an organ or tissue that is usually free ofbacteria (e.g., an organ or tissue that is free of bacteria in theabsence of trauma).

In some embodiments, the subject has or is at risk of hypotension and/orreduced blood pressure as a physical manifestation of radiation, traumaor shock.

In some embodiments, the subject has suffered reperfusion injury, e.g.,treated to restore blood circulation.

In some embodiments, the subject has or is at risk of having reducednutrient absorption, pain, nausea, diarrhea, and/or weight lossresulting from radiation, trauma or shock.

In some embodiments, the subject has necrotizing entercolitis, necroticenteritis, short bowel syndrome or short gut syndrome.

In some embodiments, the method returns the GI tract to normalhomeostasis. In some embodiments, the method improves absorption ofnutrients from the GI tract.

In some embodiments, the method reduces traumatic insult.

In some embodiments, the method reduces overstimulation of the immunesystem. In some embodiments, the method regulates the initiation ofregenerative pathways.

In some embodiments, the method promotes survival. In some embodiments,the method improves mortality.

In some embodiments, the method reduces damage to the GI tract. In someembodiments, the method improves healing of the epithelia and or thevilli in the GI tract. In some embodiments, the method returns the GItract to normal homeostasis. In some embodiments, the method reducesbacterial translocation across the GI tract. In some embodiments, themethod improves absorption of nutrients from the GI tract.

In some embodiments, the method reduces local inflammation. In someembodiments, the method reduces systemic inflammation.

In some embodiments, the method reduces pain and suffering in thesubject.

In some embodiments, the compound is administered at regular intervals.In some embodiments, the compound is administered periodically atregular intervals (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more timesevery 1, 2, 3, 4, 5, or 6 days).

In some embodiments, the compound is administered at a predeterminedinterval (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times every 1, 2,3, 4, 5, or 6 days). In some embodiments, the compound is administeredonce daily. In some embodiments, the compound is administered from about1 to about 5 times per day. In some embodiments, the compound isadministered from about 1 to about 3 times per day.

In some embodiments, the subject is treated for up to 3 weeks, 2 weeks,1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day.

In some embodiments, the subject is treated for 3 weeks, 2 weeks, 1week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day.

In some embodiments, the subject is treated within 1 hour, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours,20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or 2weeks after exposure to radiation, trauma, shock, or infection (e.g.,bacterial or viral infection).

In some embodiments, the derivatized chitosan is functionalized atbetween 18% and 30%.

In some embodiments, the molecular weight of the derivatized chitosan isfrom 50 to 150 kDa (e.g., between 50 and 125 kDa, e.g., between 60 to100 kDa, e.g., about 90 kDa).

In some embodiments, the polydispersity index of the derivatizedchitosan is from 1.0 to 2.5. In some embodiments, the polydispersityindex of the derivatized chitosan is from 1.5 to 2.0.

In one aspect, the invention features a method of treating a subject,the method comprising identifying a subject that will be exposed toradiation, trauma or shock; and prior to exposure to radiation, traumaor shock, treating the subject by administering a therapeuticallyeffective amount of a compound to the subject, wherein the compound is acompound of Formula (I):

wherein n is an integer between 20 and 6000; and each R¹ isindependently selected for each occurrence from hydrogen, acetyl,

wherein at least 25% of R¹ substituents are H, at least 1% of R¹substituents are acetyl, and at least 2% of R¹ substituents are

wherein upon administration of the compound, the compound treats,reduces the severity or delays the onset of sepsis or reduces thelikelihood of mortality, thereby prophylactically treating the subject.

In some embodiments, the permeability is a result of shock, trauma, orexposure to infection in the GI.

In some embodiments, the subject has a bacterial infection, chemicaldamage or radiation damage, e.g., resulting in leaky gut and/or damageto the GI tract. In some embodiments, the infection or damage results inleaky gut.

In some embodiments, the method reduces the severity of sepsis or asymptom thereof or decreases the likelihood of mortality from theradiation, trauma or shock relative to a subject not administered withthe compound.

In some embodiments, the subject has sepsis or a symptom of sepsisresulting from radiation, trauma or shock. In some embodiments, thesepsis is caused by leaky gut (e.g., mucosal lesions). In someembodiments, the subject is at risk of developing sepsis as a result ofexposure to radiation, trauma or shock.

In some embodiments, the radiation, trauma or shock results in reducedintegrity of the GI tract of the subject or leaky gut (e.g., mucosallesions in the GI tract) in a subject.

In some embodiments, the trauma or shock is a bacterial, viral, orfungal infection resulting in GI damage. In some embodiments, thebacterial infection is from one of the following bacteria: Salmonellaenterica serovar Typhimurium, Shigella flexneri, E. coli and P.aeruginosa.

In some embodiments, the subject has been exposed to radiation in anamount sufficient to produce 30% to 80% lethality, e.g., at 30% to 80%lethal dose, at LD30 to LIMO.

In some embodiments, the method reduces inflammation in the subject fromthe radiation, trauma or shock. In some embodiments, the methodmitigates the inflammatory response in the GI tract. In someembodiments, the method mitigates the inflammatory response and reducesmortality due to bacterial infection, bacterial translocation orchemical damage or radiation damage in the GI tract of a subjectrelative to a subject not administered with the compound.

In some embodiments, the method protects epithelial cells from bacterialinvasion. In some embodiments, the method reduces translocation ofbacteria across the GI tract, e.g., by up to 80%. In some embodiments,the compound acts through mucoadhesive substantivity (e.g., adhesion,affinity). In some embodiments, the method reduces crypt degeneration.In some embodiments, the method promotes the health of villousepithelium (e.g. reduces loss or blunting of villi).

In some embodiments, the method reduces mortality after exposure of theGI tract of a subject to ionizing radiation relative to a subject notadministered with the compound. In some embodiments, the radiation isfrom a dirty bomb, accidental nuclear incident or therapeutic radiationnot related to the treatment of cancer. In some embodiments, theradiation is targeted to therapeutic treatment requiring destruction ofthe immune system, e.g., bone marrow transplant therapy or otherelective exposure to radiation.

In some embodiments, the trauma results from exposure to a toxicchemical or poison. In some embodiments, the toxic chemical or poison isingested.

In some embodiments, the trauma results from multi-organ failure fromphysical damage and trauma to the body, burns, blast injury, systemicinfection, blood loss (hypotension), and traumatic brain injury.

In some embodiments, the shock is physical shock not resulting in awound. In some embodiments, the shock results from excessive stimuli,e.g. pathogens, commensals, injury, heat, autoantigens, tumors, necroticcells. In some embodiments, the trauma results from a commensal or apathogen translocating to an organ or tissue that is usually free ofbacteria (e.g., an organ or tissue that is free of bacteria in theabsence of trauma).

In some embodiments, the subject has or is at risk of hypotension and/orreduced blood pressure as a physical manifestation of radiation, traumaor shock.

In some embodiments, the subject has suffered reperfusion injury, e.g.,treated to restore blood circulation.

In some embodiments, the subject has or is at risk of having reducednutrient absorption, pain, nausea, diarrhea, and/or weight lossresulting from radiation, trauma or shock.

In some embodiments, the subject has necrotizing entercolitis, necroticenteritis, short bowel syndrome or short gut syndrome.

In some embodiments, the method returns the GI tract to normalhomeostasis. In some embodiments, the method improves absorption ofnutrients from the GI tract.

In some embodiments, the method reduces traumatic insult.

In some embodiments, the method reduces overstimulation of the immunesystem. In some embodiments, the method regulates the initiation ofregenerative pathways.

In some embodiments, the method promotes survival. In some embodiments,the method improves mortality.

In some embodiments, the method reduces damage to the GI tract. In someembodiments, the method improves healing of the epithelia and or thevilli in the GI tract. In some embodiments, the method returns the GItract to normal homeostasis. In some embodiments, the method reducesbacterial translocation across the GI tract. In some embodiments, themethod improves absorption of nutrients from the GI tract.

In some embodiments, the method reduces local inflammation. In someembodiments, the method reduces systemic inflammation.

In some embodiments, the method reduces pain and suffering in thesubject.

In some embodiments, the compound is administered at regular intervals.In some embodiments, the compound is administered periodically atregular intervals (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more timesevery 1, 2, 3, 4, 5, or 6 days).

In some embodiments, the compound is administered at a predeterminedinterval (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times every 1, 2,3, 4, 5, or 6 days). In some embodiments, the compound is administeredonce daily. In some embodiments, the compound is administered from about1 to about 5 times per day. In some embodiments, the compound isadministered from about 1 to about 3 times per day.

In some embodiments, the subject is treated for up to 3 weeks, 2 weeks,1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day.

In some embodiments, the subject is treated for 3 weeks, 2 weeks, 1week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day.

In some embodiments, the subject is treated within 1 hour, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours,20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or 2weeks after exposure to radiation, trauma, shock, or infection (e.g.,bacterial or viral infection).

In some embodiments, the derivatized chitosan is functionalized atbetween 18% and 30%.

In some embodiments, the molecular weight of the derivatized chitosan isfrom 50 to 150 kDa (e.g., between 50 and 125 kDa, e.g., between 60 to100 kDa, e.g., about 90 kDa).

In some embodiments, the polydispersity index of the derivatizedchitosan is from 1.0 to 2.5. In some embodiments, the polydispersityindex of the derivatized chitosan is from 1.5 to 2.0.

In one aspect, the invention features a method of reducing permeability(e.g., tissue damage) of the gastrointestinal tract of a subject, themethod comprising identifying a subject that has been exposed toradiation, trauma or shock, and treating the subject by administering atherapeutically effective amount of a compound to the subject, whereinthe compound is a compound of Formula (I):

wherein n is an integer between 20 and 6000; and each R¹ isindependently selected for each occurrence from hydrogen, acetyl,

wherein at least 25% of R¹ substituents are H, at least 1% of R¹substituents are acetyl, and atleast 2% of R¹ substituents are

wherein upon administration of the compound, the compound reducespermeability of the gastrointestinal tract of a subject, therebytreating the subject.

In some embodiments, the permeability is a result of shock, trauma, orexposure to infection in the GI.

In some embodiments, the subject has a bacterial infection, chemicaldamage or radiation damage, e.g., resulting in leaky gut and/or damageto the GI tract. In some embodiments, the infection or damage results inleaky gut.

In some embodiments, the method reduces the severity of sepsis or asymptom thereof or decreases the likelihood of mortality from theradiation, trauma or shock relative to a subject not administered withthe compound.

In some embodiments, the subject has sepsis or a symptom of sepsisresulting from radiation, trauma or shock. In some embodiments, thesepsis is caused by leaky gut (e.g., mucosal lesions). In someembodiments, the subject is at risk of developing sepsis as a result ofexposure to radiation, trauma or shock.

In some embodiments, the radiation, trauma or shock results in reducedintegrity of the GI tract of the subject or leaky gut (e.g., mucosallesions in the GI tract) in a subject.

In some embodiments, the trauma or shock is a bacterial, viral, orfungal infection resulting in GI damage. In some embodiments, thebacterial infection is from one of the following bacteria: Salmonellaenterica serovar Typhimurium, Shigella flexneri, E. coli and P.aeruginosa.

In some embodiments, the subject has been exposed to radiation in anamount sufficient to produce 30% to 80% lethality, e.g., at 30% to 80%lethal dose, at LD30 to LD80.

In some embodiments, the method reduces inflammation in the subject fromthe radiation, trauma or shock. In some embodiments, the methodmitigates the inflammatory response in the GI tract. In someembodiments, the method mitigates the inflammatory response and reducesmortality due to bacterial infection, bacterial translocation orchemical damage or radiation damage in the GI tract of a subjectrelative to a subject not administered with the compound.

In some embodiments, the method protects epithelial cells from bacterialinvasion. In some embodiments, the method reduces translocation ofbacteria across the GI tract, e.g., by up to 80%. In some embodiments,the compound acts through mucoadhesive substantivity (e.g., adhesion,affinity). In some embodiments, the method reduces crypt degeneration.In some embodiments, the method promotes the health of villousepithelium (e.g. reduces loss or blunting of villi).

In some embodiments, the method reduces mortality after exposure of theGI tract of a subject to ionizing radiation relative to a subject notadministered with the compound. In some embodiments, the radiation isfrom a dirty bomb, accidental nuclear incident or therapeutic radiationnot related to the treatment of cancer. In some embodiments, theradiation is targeted to therapeutic treatment requiring destruction ofthe immune system, e.g., bone marrow transplant therapy or otherelective exposure to radiation.

In some embodiments, the trauma results from exposure to a toxicchemical or poison. In some embodiments, the toxic chemical or poison isingested.

In some embodiments, the trauma results from multi-organ failure fromphysical damage and trauma to the body, burns, blast injury, systemicinfection, blood loss (hypotension), and traumatic brain injury.

In some embodiments, the shock is physical shock not resulting in awound. In some embodiments, the shock results from excessive stimuli,e.g. pathogens, commensals, injury, heat, autoantigens, tumors, necroticcells. In some embodiments, the trauma results from a commensal or apathogen translocating to an organ or tissue that is usually free ofbacteria (e.g., an organ or tissue that is free of bacteria in theabsence of trauma).

In some embodiments, the subject has or is at risk of hypotension and/orreduced blood pressure as a physical manifestation of radiation, traumaor shock.

In some embodiments, the subject has suffered reperfusion injury, e.g.,treated to restore blood circulation.

In some embodiments, the subject has or is at risk of having reducednutrient absorption, pain, nausea, diarrhea, and/or weight lossresulting from radiation, trauma or shock.

In some embodiments, the subject has necrotizing entercolitis, necroticenteritis, short bowel syndrome or short gut syndrome.

In some embodiments, the method returns the GI tract to normalhomeostasis. In some embodiments, the method improves absorption ofnutrients from the GI tract.

In some embodiments, the method reduces traumatic insult.

In some embodiments, the method reduces overstimulation of the immunesystem. In some embodiments, the method regulates the initiation ofregenerative pathways.

In some embodiments, the method promotes survival. In some embodiments,the method improves mortality.

In some embodiments, the method reduces damage to the GI tract. In someembodiments, the method improves healing of the epithelia and or thevilli in the GI tract. In some embodiments, the method returns the GItract to normal homeostasis. In some embodiments, the method reducesbacterial translocation across the GI tract. In some embodiments, themethod improves absorption of nutrients from the GI tract.

In some embodiments, the method reduces local inflammation. In someembodiments, the method reduces systemic inflammation.

In some embodiments, the method reduces pain and suffering in thesubject.

In some embodiments, the compound is administered at regular intervals.In some embodiments, the compound is administered periodically atregular intervals (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more timesevery 1, 2, 3, 4, 5, or 6 days).

In some embodiments, the compound is administered at a predeterminedinterval (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times every 1, 2,3, 4, 5, or 6 days). In some embodiments, the compound is administeredonce daily. In some embodiments, the compound is administered from about1 to about 5 times per day. In some embodiments, the compound isadministered from about 1 to about 3 times per day.

In some embodiments, the subject is treated for up to 3 weeks, 2 weeks,1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day.

In some embodiments, the subject is treated for 3 weeks, 2 weeks, 1week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day.

In some embodiments, the subject is treated within 1 hour, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours,20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or 2weeks after exposure to radiation, trauma, shock, or infection (e.g.,bacterial or viral infection).

In some embodiments, the derivatized chitosan is functionalized atbetween 18% and 30%.

In some embodiments, the molecular weight of the derivatized chitosan isfrom 50 to 150 kDa (e.g., between 50 and 125 kDa, e.g., between 60 to100 kDa, e.g., about 90 kDa).

In some embodiments, the polydispersity index of the derivatizedchitosan is from 1.0 to 2.5. In some embodiments, the polydispersityindex of the derivatized chitosan is from 1.5 to 2.0.

In one aspect, the invention features a method of treating a subject,the method comprising identifying a subject that has been exposed toradiation, trauma or shock and treating the subject with one of thefollowing:

-   -   a) a compound described herein, such as a polysaccharide        described herein such as a polyglucosamine, e.g., a compound of        Formula (I); or    -   b) a compound that protects the mucosal lining of the        gastrointestinal tract (GI) tract from translocation of bacteria        across the gut, wherein the radiation, trauma or shock results        in reduced integrity of the GI tract (e.g., leaky gut) of the        subject,        wherein upon administration of the compound to the subject, the        compound treats, reduces the severity or delays the onset of        sepsis or reduces the likelihood of mortality in a subject upon        administration of a therapeutically effective amount of the        compound to the subject, thereby treating the subject.

In some embodiments, the method reduces GI permeability and tissuedamage after shock or trauma or after exposure to bacteria. In someembodiments, the method reduces GI damage or improves GI integrity aftershock or trauma or after exposure to bacteria. In some embodiments, themethod reduces damage to the subject from the radiation. In someembodiments, the method reduces inflammation, e.g., systemicinflammation, in the subject from the radiation.

In some embodiments, the method mitigates the inflammatory response inthe GI tract. In some embodiments, the method reduces translocation ofbacteria by up to 80%. In some embodiments, the method mitigates thesystemic inflammatory response by reducing translocation of bacteriaacross the GI tract through mucoadhesive substantivity (e.g., adhesion,affinity) of the compound. In some embodiments, the method mitigates theinflammatory response by protecting epithelial cells from bacterialinvasion. In some embodiments, the method reduces inflammation.

In some embodiments, the method promotes healing in a subject.

In some embodiments, the method reduces the severity or delays the onsetof sepsis or reduces the likelihood of mortality in a subject relativeto a subject not administered with the compound.

In some embodiments, the method mitigates inflammation and reducesmortality due to bacterial infection, bacterial translocation orchemical damage or radiation damage in the GI tract of a subjectrelative to a subject not administered with the compound.

In some embodiments, the method reduces mortality after exposure of theGI tract of a subject to ionizing radiation relative to a subject notadministered with the compound.

In some embodiments, the method reduces crypt degeneration. In someembodiments, the method promotes the health of villous epithelium (e.g.reduces loss or blunting of villi).

In some embodiments, the source of radiation is a dirty bomb, accidentalnuclear incident or therapeutic radiation, e.g. other than that relatedto the treatment of cancer. In some embodiments, the radiation causesphysiologic changes in the GI tract. In some embodiments, the subjecthas been exposed to radiation in an amount sufficient to produce 30% to80% lethality, e.g., at 30% to 80% lethal dose, at LD30 to LD80.

In some embodiments, the source of radiation is targeted to therapeutictreatment requiring destruction of the immune system, e.g., bone marrowtransplant therapy or other elective exposure to radiation. In someembodiments, the therapeutic treatment causes the GI tract to becomeleaky or to lose integrity.

In some embodiments, the source of the trauma is exposure to a toxicchemical or poison. In some embodiments, the toxic chemical or poison isingested.

In some embodiments, the source of the trauma is multi-organ failure dueto accepted modes of failure, e.g., physical damage and trauma to thebody, burns, blast injury, systemic infection, blood loss (hypotension),and traumatic brain injury.

In some embodiments, the source of shock is physical shock not resultingin a wound. In some embodiments, the source of shock is excessivestimuli, e.g. pathogens, commensals, injury, heat, autoantigens, tumors,necrotic cells. In some embodiments, the trauma results from a commensalor a pathogen translocating to an organ or tissue that is usually freeof bacteria (e.g., an organ or tissue that is free of bacteria in theabsence of trauma).

In some embodiments, the subject has a bacterial infection, chemicaldamage or radiation damage in the GI tract. In some embodiments, thesubject has been exposed to sufficient radiation to cause a leaky GItract or mucosal lesions. In some embodiments, the subject has beenexposed to radiation in an amount sufficient to produce 30% to 80%lethality, e.g., at 30% to 80% lethal dose, at LD30 to LD80.

In some embodiments, the subject has leaky gut, wherein the leaky gut isa result of exposure to radiation, trauma or shock.

In some embodiments, the subject has sepsis or a symptom of sepsisresulting from radiation, trauma or shock. In some embodiments, thesepsis is caused by leaky gut. In some embodiments, the subject is atrisk of developing sepsis as a result of exposure to radiation, traumaor shock.

In some embodiments, the subject has or is at risk of hypotension, e.g.,reduced blood pressure as a physical manifestation of radiation, traumaor shock. In some embodiments, the subject has suffered reperfusioninjury, e.g., treated to restore blood circulation.

In some embodiments, the subject has or is at risk of having reducednutrient absorption, pain, nausea, diarrhea, and/or weight lossresulting from radiation, trauma or shock.

In some embodiments, the subject has necrotizing entercolitis, necroticenteritis, short bowel syndrome or short gut syndrome.

In some embodiments, the subject has a bacterial infection wherein thebacteria is Salmonella enterica serovar Typhimurium, Shigella flexneri,E. coli or P. aeruginosa.

In some embodiments, the compound is a polyglucosamine. Exemplarypolyglucosamines include those soluble at acid, physiological pH or morebasic pH (e.g., the pH of the digestive tract), e.g., the pH of theintestine (e.g., small intestine or large intestine) or colon; and or acharged polyglucosamine, e.g., poly (acetyl, arginyl) glucosamine(PAAG). In some embodiments, the polyglucosamine is a chitosan, e.g., achitosan soluble at physiological pH.

In some embodiments, the soluble polyglucosamine comprises apolyglucosamine of the following Formula (I):

wherein n is an integer between 20 and 6000; and each R¹ isindependently selected for each occurrence from hydrogen, acetyl, andeither a) a group of Formula (II):

wherein R² is hydrogen or amino; and R³ is amino, guanidino, C₁-C₆ alkylsubstituted with an amino or guanidino moiety, or a natural or unnaturalamino acid side chain; or b) R¹, when taken together with the nitrogento which it is attached, forms a guanidine moiety;wherein at least 25% of R¹ substituents are H, at least 1% of R¹substituents are acetyl, and at least 2% of R¹ substituents are a groupof Formula (II) or are taken together with the nitrogen to which theyare attached to form a guanidine moiety.

In some embodiments, the soluble polyglucosamine comprises apolyglucosamine of the following Formula (I), wherein at least 90% bynumber or weight of R¹ moieties are as defined in Formula (I) (e.g., atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99%):

wherein n is an integer between 20 and 6000; and each R¹ isindependently selected for each occurrence from hydrogen, acetyl, andeither a) a group of Formula (II):

wherein R² is hydrogen or amino; and R³ is amino, guanidino, C₁-C₆ alkylsubstituted with an amino or guanidino moiety, or a natural or unnaturalamino acid side chain; or b) R¹, when taken together with the nitrogento which it is attached, forms a guanidine moiety; wherein at least 25%of R¹ substituents are H, at least 1% of R¹ substituents are acetyl, andat least 2% of R¹ substituents are a group of Formula (II) or are takentogether with the nitrogen to which they are attached to form aguanidine moiety.

In some embodiments, between 25-95% of R¹ substituents are hydrogen. Insome embodiments, between 55-90% of R¹ substituents are hydrogen.

In some embodiments, between 1-50% of R¹ substituents are acetyl. Insome embodiments, between 4-20% of R¹ substituents are acetyl.

In some embodiments, between 2-50% of R¹ substituents are a group ofFormula (II). In some embodiments, between 4-30% of R¹ substituents area group of Formula (II).

In some embodiments, 55-90% of R¹ substituents are hydrogen, 4-20% of R¹substituents are acetyl, 4-30% of R¹ substituents are a group of Formula(II).

In some embodiments, R² is amino and R³ is an arginine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino and R³ is a lysine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, R² is amino and R³ is a histidine side chain.

In some embodiments, R¹ is selected from one of the following:

In some embodiments, at least 1% of R¹ substituents are selected fromone of the following:

and at least 1% of R¹ substituents are selected from the following:

In some embodiments, R² is amino and R³ is a substituted C₁-C₆ alkyl.

In some embodiments, R³ is C₁-C₆ alkyl substituted with an amino group.In some embodiments, R³ is C₁ alkyl substituted with an amino group. Insome embodiments, R³ is C₂ alkyl substituted with an amino group. Insome embodiments, R³ is C₃ alkyl substituted with an amino group. Insome embodiments, R³ is C₄ alkyl substituted with an amino group. Insome embodiments, R³ is C₅ alkyl substituted with an amino group. Insome embodiments, R³ is C₆ alkyl substituted with an amino group. Insome embodiments, R¹ is selected from one of the following:

In some embodiments, R³ is C₁-C₆ alkyl substituted with a guanidinogroup. In some embodiments, R³ is C₁ alkyl substituted with a guanidinogroup. In some embodiments, R³ is C₂ alkyl substituted with a guanidinogroup. In some embodiments. R³ is C₃ alkyl substituted with a guanidinogroup. In some embodiments, R³ is C₄ alkyl substituted with a guanidinogroup. In some embodiments, R³ is C₅ alkyl substituted with a guanidinogroup. In some embodiments, R³ is C₆ alkyl substituted with a guanidinogroup. In some embodiments, R¹ is selected from one of the following:

In some embodiments, wherein R² is amino that is substituted with anitrogen protecting group prior to substitution (e.g.,functionalization) on the polyglucosamine and removed subsequent tosubstitution (e.g., functionalization) on the polyglucosamine.

In some embodiments, the nitrogen protecting group istert-butyloxycarbonyl (Boc).

In some embodiments, a nitrogen protecting group is used in thesynthetic process, which can provide an intermediate polymer having anitrogen protecting group such as Boc.

In some embodiments, R² is amino.

In some embodiments, R² is hydrogen and R³ is amino.

In some embodiments, R² is hydrogen and R³ is guanidino.

In some embodiments, R² is hydrogen and R³ is a substituted C₁-C₆ alkyl.In some embodiments, R³ is C₁-C₆ alkyl substituted with an amino group.In some embodiments, R³ is C₁ alkyl substituted with an amino group. Insome embodiments, R³ is C₂ alkyl substituted with an amino group. Insome embodiments, R³ is C₃ alkyl substituted with an amino group. Insome embodiments, R³ is C₄ alkyl substituted with an amino group. Insome embodiments, R³ is C₅ alkyl substituted with an amino group. Insome embodiments, R³ is C₆ alkyl substituted with an amino group. Insome embodiments, R¹ is selected from one of the following:

In some embodiments, R³ is C₁-C₆ alkyl substituted with a guanidinogroup. In some embodiments, R³ is C₁ alkyl substituted with a guanidinogroup. In some embodiments, R³ is C₂ alkyl substituted with a guanidinogroup. In some embodiments, R³ is C₃ alkyl substituted with a guanidinogroup. In some embodiments, R³ is C₄ alkyl substituted with a guanidinogroup. In some embodiments, R³ is C₅ alkyl substituted with a guanidinogroup. In some embodiments, R³ is C₆ alkyl substituted with a guanidinogroup. In some embodiments, R¹ is selected from one of the following:

In some embodiments, at least 25% of R¹ substituents are H, at least 1%of R¹ substituents are acetyl, and at least 2% of R¹ substituents areindependently selected from any of the formulae specifically shownabove.

In some embodiments, the functionalized polyglucosamine of Formula (I)may be further derivatized (i.e., functionalized) on the free hydroxylmoieties.

In some embodiments, the molecular weight of the functionalizedpolyglucosamine is between Sand 1,000 kDa. In some embodiments, themolecular weight of the functionalized polyglucosamine is between 10 and350 kDa. In some embodiments, the molecular weight of the functionalizedpolyglucosamine is between 15 and 200 kDa. In some embodiments, themolecular weight of the functionalized polyglucosamine is between 25 and175 kDa. In some embodiments, the molecular weight of the functionalizedpolyglucosamine is between 50 and 150 kDa (e.g., between 50 and 125 kDa,e.g., between 60 to 100 kDa, e.g., about 90 kDa).

In some embodiments, the functionalized polyglucosamine is soluble inaqueous solution between pH 3 and 11. In some embodiments, thefunctionalized polyglucosamine is soluble in aqueous solution between pH2 and 10. In some embodiments, the functionalized polyglucosamine issoluble in aqueous solution between pH 5 and 9. In some embodiments, thefunctionalized polyglucosamine is soluble in aqueous solution between pH6.8 and pH 7.4.

In some embodiments, the functionalized polyglucosamine is soluble inaqueous solution at all physiological pH ranges.

In some embodiments, the functionalized polyglucosamine is soluble inaqueous solution at all pH ranges of the gastrointestinal tract, e.g.,pH 3.

In some embodiments, the functionalized polyglucosamine is soluble inaqueous solution at all pH ranges of the intestinal tract, e.g., smallintestine, e.g., at up to pH 8.

In some embodiments, the polyglucosamine is functionalized at between 5%and 50%. In some embodiments, the polyglucosamine is functionalized atbetween 15% and 35%.

In some embodiments, the degree of deacetylation (% DDA) of thederivatized (i.e., functionalized) polyglucosamine is between 75% and99%. In some embodiments, the degree of deacetylation (% DDA) of thederivatized (i.e., functionalized) polyglucosamine is between 80% and98%.

In some embodiments, the polydispersity index (PDT) of the derivatized(i.e., functionalized) polyglucosamine is between 1.0 and 2.5. In someembodiments, the polydispersity index (PDI) of the derivatized (i.e.,functionalized) polyglucosamine is between 1.2 and 1.8.

In some embodiments, the functionalized polyglucosamine is substantiallyfree of other impurities.

In some embodiments, the compound is administered orally.

In some embodiments, the compound is administered by enema.

In some embodiments, the compound is administered to the GI tract of thesubject.

In some embodiments, the compound is administered to the subject after24 hours of the subject's exposure to radiation, trauma or shock.

In some embodiments, the compound is administered to the subject within24 hours of the subject's exposure to radiation, trauma or shock.

In some embodiments, the compound is administered to the subject priorto the subject's exposure to radiation, trauma or shock.

In some embodiments, the compound is administered in a therapeuticallyeffective amount. In some embodiments, the therapeutically effectiveamount is up to 100 mg/kg. In another embodiment, the effective amountis 5 to 500 mg/kg. In another embodiment, the effective amount is 1 to50 mg/kg. In another embodiment, the effective amount is 5 to 50 mg/kgwith additional dosing of 100 to 500 μg/ml in solution.

In another embodiment, the therapeutically effective amount is up to 100ug/kg.

In some embodiments, the compound is administered for 1 day, 2 days, 4days, 1 week, 2 weeks, 4 weeks or until symptoms cease.

In another embodiment, the effective amount is 40 μg/kg thrice daily.

In some embodiments, the treatment reduces traumatic insult. In someembodiments, the treatment reduces overstimulation of the immune system.In some embodiments, the treatment regulates the initiation ofregenerative pathways. In some embodiments, the treatment promotessurvival. In some embodiments, the treatment reduces damage to the GItract. In some embodiments, the treatment improves healing of theepithelia and or the villi in the GI tract. In some embodiments, thetreatment improves mortality. In some embodiments, treatment returns theGT tract to normal homeostasis. In some embodiments, the treatmentreduces bacterial translocation across the GI tract. In someembodiments, the treatment improves absorption of nutrients from the GItract.

In some embodiments, the treatment reduces local inflammation. In someembodiments, the treatment reduces systemic inflammation.

In some embodiments, the treatment reduces the World Health Organization(WHO) mucositis score. In some embodiments, the method reduces thepercentage of animals with a WHO mucositis score greater than or equalto 3. In some embodiments, the method reduces ulceration 10% or more ina subject.

In one aspect, the invention features a method of prophylactic treatmentof a subject, the method comprising identifying a subject that will beexposed to radiation, trauma or shock; and prior to exposure toradiation, trauma or shock, treating the subject with one of thefollowing:

-   -   a) a compound described herein, such as a polysaccharide        described herein such as a polyglucosamine, e.g., a compound of        Formula (I); or    -   b) a compound that protects the mucosal lining of the        gastrointestinal tract (GI) tract from translocation of bacteria        across the gut, wherein the radiation, trauma or shock results        in reduced integrity of the GI tract (e.g., leaky gut) of the        subject,        wherein upon administration of the compound to the subject, the        compound treats, reduces the severity or delays the onset of        sepsis or reduces the likelihood of mortality in a subject upon        administration of a therapeutically effective amount the        compound to the subject, thereby prophylactically treating the        subject.

In one aspect, the invention features a method of reducing the severityor delaying the onset of sepsis or decreasing the likelihood ofmortality of a subject that has been exposed to radiation, trauma orshock; the method comprising administering to the subject one of thefollowing:

-   -   a) a compound described herein, such as a polysaccharide        described herein such as a polyglucosamine, e.g., a compound of        Formula (I); or    -   b) a compound that protects the mucosal lining of the        gastrointestinal tract (GI) tract from translocation of bacteria        across the gut, wherein the radiation, trauma or shock results        in reduced integrity of the GI tract (e.g., leaky gut) of the        subject,        wherein upon administration of the compound to the subject, the        compound reduces the severity or delays the onset of sepsis or        reduces the likelihood of mortality in a subject upon        administration of a therapeutically effective amount the        compound to the subject, thereby treating the subject.

In one aspect, the invention features a method of reducing the severityor delaying the onset of sepsis or decreasing the likelihood ofmortality of a subject that will be exposed to radiation, trauma orshock; the method comprising prophylactic administration to the subjectone of the following:

-   -   a) a compound described herein, such as a polysaccharide        described herein such as a polyglucosamine, e.g., a compound of        Formula (I); or    -   b) a compound that protects the mucosal lining of the        gastrointestinal tract (GI) tract from translocation of bacteria        across the gut, wherein the radiation, trauma or shock results        in reduced integrity of the GI tract (e.g., leaky gut) of the        subject,        wherein upon administration of the compound to the subject, the        compound reduces the severity or delays the onset of sepsis or        reduces the likelihood of mortality in a subject upon        prophylactic administration of a therapeutically effective        amount the compound to the subject, thereby treating the        subject.

In one aspect, the invention features a method of treating mucositis(e.g., in the gastrointestinal (GI) tract) in a subject exposed toradiation, trauma or shock, the method comprising identifying a subjectthat has been exposed to radiation, trauma or shock; and treating thesubject with one of the following:

-   -   a) a compound described herein, such as a polysaccharide        described herein such as a polyglucosamine, e.g., a compound of        Formula (I); or    -   b) a compound that protects the mucosal lining of the GI tract        from translocation of bacteria across the gut, wherein the        radiation, trauma or shock results in reduced integrity of the        GI tract (e.g., leaky gut) of the subject,        wherein upon treating the subject with a therapeutically        effective amount of the compound to the subject, the compound        treats mucositis in a subject that has been exposed to        radiation, trauma or shock, thereby treating the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an exemplary effect of PAAG and radiation treatment onsurvival.

FIG. 2 depicts an exemplary effect of PAAG and radiation treatment onweight.

FIG. 3 depicts an exemplary comparison of mean oral mucositis scores fora vehicle control and four doses of PAAG.

FIG. 4 depicts an exemplary comparison of weight change for a vehiclecontrol and four doses of PAAG given as an oral rinse.

FIG. 5 depicts an exemplary comparison of the percentage of animals withan oral mucositis score of 3 or higher on a given day post irradiationfor a vehicle control and two doses of PAAG.

FIG. 6 depicts an exemplary comparison of the percentage of animals witha mucositis score of 3 or higher on a given day post irradiation for avehicle control and three doses of PAAG.

FIG. 7 depicts an exemplary comparison of the mean mucositis scores fora vehicle control and 200 ppm PAAG administered thrice daily at fourdifferent time windows.

FIG. 8 depicts an exemplary comparison of the mean percent weight gainfor a vehicle control and 200 ppm PAAG administered thrice daily at fourdifferent scheduling windows.

FIG. 9 depicts an exemplary comparison of the percentage of animals witha score of 3 or greater for a vehicle control and 200 ppm PAAGadministered thrice daily at two different scheduling windows.

FIG. 10 depicts an exemplary comparison of the production of IL8 in U937cells following stimulation with LPS with or without pretreatment with100 ppm PAAG or lactoferrin.

FIG. 11 depicts an exemplary comparison of binding of MRSA to nasalepithelial cells following pretreatment with media, 200 or 500 ppm PAAG.

FIG. 12A depicts an exemplary comparison of attachment of Acinetobacterbaumannii into CaCo2 cells following various pretreatments of the cellsby vehicle or 200 ppm PAAG. FIG. 12B depicts the comparison of invasionof Burkholderia cepacia complex into CaCo2 cells following variouspretreatments of the cells by vehicle or 200 ppm PAAG.

FIG. 13 depicts exemplary A431 epidermal cells, scratched and treatedfor 24, 48, 72 or 96 hours with nothing, PAAG (here noted as CA), EGF orPAAG (CA)+EGF. The circle on top right is a bubble.

FIG. 14 depicts an exemplary necrotic enteritis model of pathogenicinfection showing % mortality of chicks 14 days after exposure to lethaldoses of C. perfringens with coccidian sensitization. Bacitracin and 100ppm PAAG delivered ad libitum in water reduced mortality withstatistical significance relative to control. 10 ppm was numericallysignificant vs. control.

FIG. 15 depicts exemplary photomicrographs of selected mice at 4 dayspost-irradiation, with 13 Gy-radiation without treatment (Animal 54) andwith PAAG treatment (Animal 67).

FIG. 16 depicts exemplary total bacterial colony forming units (CFU) pergram of mesenteric lymph nodes in control or irradiated mice treatedwith vehicle or PAAG once per day via oral gavage at day 2, 3 and 4starting 24 hours after TBI.

FIG. 17 depicts exemplary inflammation, epithelial loss, crypt loss, andcrypt regeneration on day 2

FIG. 18 depicts exemplary inflammation, crypt loss, and cryptregeneration on day 3.

FIG. 19 depicts exemplary inflammation, epithelial loss, crypt loss, andcrypt regeneration on day 4.

FIG. 20 depicts an exemplary increase in inflammation, epithelial loss,crypt loss, and crypt regeneration over time after radiation exposure.

FIG. 21 depicts exemplary inflammation, epithelial loss, crypt loss, andcrypt regeneration over time in different sections of gut afterradiation exposure.

FIG. 22 depicts exemplary photomicrographs of small intestines ofanimals in Group 1 (untreated+no radiation).

FIG. 23 depicts exemplary photomicrographs of small intestines ofanimals in Group 2 (PAAG+no radiation).

FIG. 24 depicts exemplary photomicrographs at 2 days post-irradiation ingroup 3 animals (vehicle+13 Gy radiation).

FIG. 25 depicts exemplary photomicrographs at 4 days post-irradiationanimals in group 3 (vehicle+13 Gy radiation).

FIG. 26 depicts exemplary photomicrographs at 2 days post-irradiationanimals in group 4 (PAAG+13 Gy radiation).

FIG. 27 depicts exemplary photomicrographs at 4 days post-irradiationanimals in group 4 (PAAG+13 Gy radiation).

FIG. 28 depicts exemplary Pro-calcitonin (PCT) in plasma for micetreated and untreated with radiation and PAAG.

FIG. 29 depicts exemplary citrulline levels in plasma for mice treatedand untreated with radiation and PAAG.

FIG. 30 depicts an exemplary effect of PAAG and radiation treatment onsurvival.

DETAILED DESCRIPTION

Described herein are methods of treating a subject, for example, eitherprior to or subsequent to when the subject has been exposed to orexperienced radiation, trauma, or shock. In some embodiments, themethods of treating a subject can help treat and/or prevent leaky gut ina subject. In some embodiments, the methods of treating a subject canhelp prevent the subject from experiencing sepsis, or reduce theseverity of the sepsis. This can be achieved by administering to thesubject a compound described herein. Examples of such compounds includepolysaccharides, including polyglucosamines such as those describedherein (e.g., a compound of Formula (I)). In some embodiments, thetreatment can reduce the severity of a symptom or decrease thelikelihood of mortality of a subject that has been or will be exposed toradiation, trauma or shock. In some embodiments, the compound protectsthe mucosal lining of the GI tract from allowing bacterial translocationacross the gut. In some embodiments, the compound inhibits binding ofbacteria or toxins, endotoxins, mitochondria, pathogen associatedmolecular patterns (PAMPs), damage associated molecular patterns(DAMPs), and chemotherapy and radiation associated molecular patterns(CRAMPs) in the subject with an innate immune receptor such as Toll-likeReceptor 4 (TLR4). In some embodiments, the compound reducesTNF-expression in response to Lipopolysaccharide (LPS). In someembodiments, the compound reduces IL-8 production in response to PAMP orCRAMP stimuli. In some embodiments, the compound inhibits bacterialadhesion to cells of the mucosal lining of the GI tract of the subject.

Treatment

The compositions and compounds described herein (e.g., a compound thatprotects the mucosal lining or integrity of the GI tract, such as asoluble polyglucosamine or a derivatized (i.e., functionalized)polyglucosamine described herein) can be administered to a tissue, e.g.in vitro or ex vivo, or to a subject, e.g., in vivo, to treat and/orprevent a variety of conditions resulting from radiation, trauma orshock, including those described herein below.

As used herein, the term “treat” or “treatment” is defined as theapplication or administration of a composition or compound (e.g., acompound described herein (e.g., a compound that protects the mucosallining of the GI tract such as a soluble or derivatized (i.e.,functionalized) polyglucosamine described herein)) to a subject, e.g., apatient, or application or administration of the composition or compoundto an isolated tissue, from a subject, e.g., a patient, who has beenexposed to radiation, trauma or shock, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve and/or affect thesubject, one or more symptoms of a disorder described herein or thepredisposition toward a disorder described herein (e.g., to prevent atleast one symptom of the disorder described herein and/or to delay onsetof at least one symptom of the disorder described herein), and/or a sideor adverse effect of radiation, trauma or shock. A subject issuccessfully “treated” if, after receiving an effective amount of one ormore active agents described herein, the subject shows observable and/ormeasurable reduction in or absence of morbidity and/or mortality,improvement in nutrient adsorption, reduction of pain, reduction ofnausea, reduction of diarrhea, reduction in weight loss and/orimprovement in quality of life issues.

As used herein, the term “prevent” or “prevention” is defined as theapplication or administration of a composition or compound (e.g., acompound described herein (e.g., a soluble or derivatized (i.e.,functionalized) polyglucosamine)) to a subject, e.g., a subject who isat risk for a disorder (e.g., a disorder described herein), or has adisposition toward a disorder described herein, or application oradministration of the compound to an isolated tissue from a subject,e.g., a subject who is at risk for a disorder (e.g., a disorder asdescribed herein), or has a predisposition toward a disorder describedherein, with the purpose to avoid or preclude the disorder describedherein, or affect the predisposition toward the disorder describedherein (e.g., to prevent at least one symptom of the disorder describedherein or to delay onset of at least one symptom of the disorderdescribed herein). “Preventing” a disease may also be referred to as“prophylaxis” or “prophylactic treatment.”

As used herein, an amount of a composition or compound effective totreat a disorder described herein, or a “therapeutically effectiveamount” refers to an amount of the composition or compound which iseffective, upon single or multiple dose administration to a subject, intreating a tissue, or in curing, alleviating, relieving or improving asubject with a disorder described herein beyond that expected in theabsence of such treatment.

As used herein, an amount of a composition or compound effective toprevent a disorder described herein, or “a prophylactically effectiveamount” of the composition or compound refers to an amount effective,upon single- or multiple-dose administration to the subject, inpreventing or delaying the occurrence of the onset or recurrence of adisorder described herein or a symptom of the disorder described herein.Typically, because a prophylactic dose is used in subjects prior to orat an earlier stage of disease, the prophylactically effective amountwill be less than the therapeutically effective amount.

As used herein, “administered in combination” or a combinedadministration of two agents means that two or more agents (e.g.,compounds described herein) are administered to a subject at the sametime or within an interval such that there is overlap of an effect ofeach agent on the patient. Preferably they are administered within 60,30, 15, 10, 5, or 1 minute of one another. Preferably theadministrations of the agents are spaced sufficiently close togethersuch that a combinatorial (e.g., a synergistic) effect is achieved. Thecombinations can have synergistic effect when used to treat a subjecthaving a bacterial infection. The agents can be administeredsimultaneously, for example in a combined unit dose (providingsimultaneous delivery of both agents). Alternatively, the agents can beadministered at a specified time interval, for example, an interval ofminutes, hours, days or weeks. Generally, the agents are concurrentlybioavailable, e.g., detectable, in the subject. Alternately, the solublepolyglucosamine or polyglucosamine derivative can be administeredtopically, intranasally, via pulmonary aerosol or orally, and the secondagent can be administered systemically.

In an embodiment, the agents are administered essentiallysimultaneously, for example two unit dosages administered at the sametime, or a combined unit dosage of the two agents. In anotherembodiment, the agents are delivered in separate unit dosages. Theagents can be administered in any order, or as one or more preparationsthat includes two or more agents. Alternatively, the second agent can beadministered systemically and can be available systemically during theadministration of the first agent. In an embodiment, at least oneadministration of one of the agents, e.g., the first agent, is madewithin minutes, one, two, three, or four hours, or even within one ortwo days of the other agent, e.g., the second agent. In some cases,combinations can achieve synergistic results, e.g., greater thanadditive results, e.g., at least 1.25, 1.5, 2, 4, 10, 20, 40, or 100,1000, 100000, or 100000 times greater than additive.

Trauma can be an injury e.g., a physical injury or insult, caused by anexternal source, e.g., from a fracture or blow. Trauma is the sixthleading cause of death worldwide, accounting for 10% of all mortalities,and is therefore a serious public health problem with significant socialand economic costs. Trauma can lead to significant and rapid blood loss,which can result in phyisological shock. Trauma can also be a result ofinfection, e.g., bacterial, viral, or fungal infection.

Shock is a failure of the circulatory system to supply sufficient bloodto peripheral tissues to meet basic needs including metabolicrequirements for oxygen and nutrients and incomplete removal ofmetabolic wastes from the affected tissues. Shock is usually caused byhemorrhage or overwhelming infection and is characterized in most casesby a weak, rapid pulse; low blood pressure; and cold, sweaty skin. Shockmay result from a variety of physiological mechanisms, including suddenreductions in the total blood volume such as severe hemorrhage; suddenreductions in cardiac output, as in myocardial infarction (heartattack); and widespread dilation of the blood vessels, as in some formsof infection. Typical signs of shock include low blood pressure, a rapidheartbeat and signs of poor end-organ perfusion or “decompensation”(such as low urine output, confusion or loss of consciousness). However,a subject's blood pressure may also remain stable and still be incirculatory shock. Shock can include circulatory shock, which can be alife-threatening medical condition that occurs due to inadequatesubstrate for aerobic cellular respiration. Circulatory shock can be alife-threatening medical emergency and one of the most common causes ofdeath for critically ill people. Shock can have a variety of effects,all with similar outcomes, but all relate to a problem with the body'scirculatory system. For example, shock may lead to hypoxemia (a lack ofoxygen in arterial blood) or cardiac arrest.

Subject

The subject can be a human or a non-human animal. Suitable humansubjects includes, e.g., a human patient that has been or will beexposed to radiation, trauma or shock or damage as a result of exposureto radiation, trauma or shock described herein or a normal subject. Theterm “non-human animals” of the invention includes all vertebrates,e.g., non-mammals (such as chickens, amphibians, reptiles) and mammals,such as non-human primates, e.g., elephant, sheep, dog, cat, cow, andpig. Suitable animal subjects include: but are not limited to, wildanimals, farm animals, zoo animals, circus animals, companion (pet)animals, domesticated and/or agriculturally useful animals. Suitableanimal subjects include primates, rodents, and birds. Examples of saidanimals include, but are not limited to, elephants, guinea pigs,hamsters, gerbils, rat, mice, rabbits, dogs, cats, horses, pigs, sheep,cows, goats, deer, rhesus monkeys, monkeys, tamarinds, apes, baboons,gorillas, chimpanzees, orangutans, gibbons, fowl, e.g., pheasant, quail(or other gamebirds), waterfowl, ostriches, chickens, turkeys, ducks,and geese or free flying bird.

In some embodiments, the subject has been exposed to radiation, e.g.,from a dirty bomb, accidental nuclear incident or therapeutic radiation,e.g. other than that related to the treatment of cancer. In someembodiments, the subject has been exposed to a chemical, biological orradiological agent, or has suffered chemical, biological, orradiological injury. In some embodiments, the source of the trauma isexposure to a toxic chemical or poison. In some embodiments, the toxicchemical or poison is ingested. In some embodiments, the subject is atrisk for sepsis and/or death resulting from the radiation, trauma orshock.

In some embodiments, the source of radiation is targeted to therapeutictreatment requiring destruction of the immune system, e.g., bone marrowtransplant therapy or other elective exposure to radiation. In someembodiments, the subject has been exposed to radiation in an amountsufficient to produce 30% to 80% lethality, e.g., at 30% to 80% lethaldose, at LD30 to LD80. In some embodiments, the radiation is 12 to 15 Gyradiation.

In some embodiments, the source of the trauma is multi-organ failure dueto accepted modes of failure, e.g., physical damage and trauma to thebody, burns, blast injury, systemic infection, blood loss (hypotension),traumatic brain injury.

Sepsis

Methods of treating a subject who has sepsis or displays symptoms ofsepsis are described herein. In some embodiments, the subject is at riskof sepsis as a result of exposure to radiation, trauma or shock. Sepsiscan result from septicemia (i.e., organisms, their metabolicend-products or toxins in the blood stream), including bacteremia (i.e.,bacteria in the blood), as well as toxemia (i.e., toxins in the blood),including endotoxemia (i.e., endotoxin in the blood). The term“bacteremia” includes occult bacteremia observed in young febrilechildren with no apparent foci of infection. The term “sepsis” alsoencompass that caused by fungemia (i.e., fungi in the blood), viremia(i.e., viruses or virus particles in the blood), and parasitemia (i.e.,helminthic or protozoan parasites in the blood). Gram-negative sepsis isa common type of sepsis and is caused by Escherichia coli, Klebsiellapneumonia and Pseudomonas aeruginosa. Gram-positive pathogens such asthe Staphylococci and Streptococci also causes of sepsis. A third majorgroup that causes sepsis includes fungi.

Phenotypes associated with septicemia and septic shock (acutecirculatory failure resulting from septicemia often associated withmultiple organ failure and a high mortality rate) are symptoms ofsepsis. Symptoms of sepsis in a subject include but are not limited to,increased respiration, increased heart rate, reduced arterial CO₂saturation, arterial hypotension, metabolic acidosis, fever, decreasedsystemic vascular resistance, tachypnea and organ dysfunction (asmanifest by, but not limited to, elevated transaminase, creatinine, andblood urea nitrogen).

Chemical Warfare Agents and Injury

Methods of treating a subject who has been exposed to a chemical warfareagent or has suffered a chemical warfare injury are described herein.Chemical agents that can cause chemical injury in a subject and/or beused as a chemical warfare agent include, e.g., harassing agents (e.g.,tear agents or lachrymatory agents (e.g., α-chlorotoluene, benzylbromide, bromoacetone (BA), bromobenzylcyanide (CA), bromomethyl ethylketone, capsaicin (OC), chloracetophenone (MACE; CN), chloromethylchloroformate, dibenzoxazepine (CR), ethyl iodoacetate,ortho-chlorobenzylidene malononitrile (super tear gas; CS),trichloromethyl chloroformate, and xylyl bromide), vomiting agents(e.g., adamsite (DM), diphenylchloroarsine (DA), diphenylcyanoarsine(DC))), incapacitating agents (e.g., psychological agents (e.g.,3-quinuclidinyl benzilate (BZ), phencyclidine (SN), lysergic aciddiethylamide (K)), KOLOKOL-1 (tranquilizer)), lethal agents (e.g.,blister agents (e.g., vesicants (e.g., nitrogen mustards (e.g.,bis(2-chloroethyl)ethylamine (HN1), bis(2-chloroethyl)methylamine (HN2),tris(2-chloroethyl)amine (HN3)), sulfur mustards (e.g.,1,2-bis(2-chloroethylthio) ethane (Sesquimustard; Q),1,3-bis(2-chloroethylthio)-n-propane,1,4-bis(2-chloroethylthio)-n-butane,1,5-bis(2-chloroethylthio)-n-pentane, 2-chloroethylchloromethylsulfide,bis(2-chloroethyl) sulfide (mustard gas; HD), bis(2-chloroethylthio)methane, bis(2-chloroethylthiomethyl) ether, bis(2-chloroethylthioethyl)ether (0 mustard; T)), arsenicals (e.g., ethyldichloroarsine (ED),methyldichloroarsine (MD), phenyldichloroarsine (PD),2-chlorovinyldichloroarsine (Lewisite; L))), urticants (e.g., phosgeneoxime (CX))), blood agents (e.g., cyanogen chloride (CK), hydrogencyanide (AC), arsine (SA)), choking agents or pulmonary agents (e.g.,chlorine (CL), chloropicrin (PS), diphosgene (DP), phosgene (CG)), nerveagents (e.g., G series (e.g., tabun (GA), sarin (GB), soman (GD),cyclosarin (GF)), GV series (e.g., novichok agents, GV (nerve agent)), Vseries (e.g., VE, VG, VM, VX)).

Leaky Gut

Methods of treating a subject who has leaky gut or symptoms of leaky gutare described herein. Leaky gut generally refers to intestinal or bowelhyperpermeability. The condition can allow toxins, bacteria, and foodparticles penetrate the lining of the intestinal tract and enter thebody's blood stream. Leaky gut can refer to an acute condition resultingfrom exposure or insult (e.g., from radiation, trauma, shock, orinfection (e.g., bacterial, viral, fungal infection). Leaky gut canresult in sepsis. Acute conditions are generally severe and sudden inonset (e.g., a broken bone, an asthma attack). Leaky gut can lead to acondition of an altered or damaged bowel lining (e.g., mucosa of theintestinal tract is compromised) that is caused by increasedpermeability of the gut wall from e.g., toxins, poor diet, parasites,infection, or mediations. In some embodiments, the methods describedherein treat an acute condition that can result in or be a symptom ofleaky gut.

Research and clinical diagnostic tests are available but typically notrelied upon for diagnosis of leaky gut. Probes of intermediate molecularweight (e.g., 150-400 g/mol, e.g., Cr EDTA, PEG 400, lactulose,mannitol, rhamnose) can be used to measure intestinal permeability andfor analyzing urinary recovery. Measurement of the translocation oflipopolysaccharide molecules across the gut wall also may be used tocharacterize leaky gut.

Compounds for Treating or Prophylactically Treating a Subject

Methods for treating or prophylactically treating damage resulting fromradiation, trauma or shock with a polyglucosamine compound orcomposition are described herein.

Soluble Polyglucosamines and Polyglucosamines Derivatives

The compounds described herein include polyglucosamines andpolyglucosamine derivatives. Exemplary polyglucosamines includepolyglucosamine compounds such as chitosan, e.g., a chitosan soluble inphysiological pH.

Polyglucosamines can be derived from chitosan by deacetylation. Chitosanis an insoluble polymer derived from chitin, which is a polymer ofN-acetylglucosamine that is the main component of the exoskeletons ofcrustaceans (e.g. shrimp, crab, lobster). Polyglucosamines are alsofound in various fungi and arthropods. Synthetic sources and alternatesources of β1-4 polyglucosamines may serve as the starting material forthe polyglucosamine derivatives. The polyglucosamine derivativesdescribed herein are generated by functionalizing the free amino groupswith positively charged or neutral moieties, as described herein. Up to50% of the amino groups are acetylated. For the purposes of thisinvention, if greater than 50% of the amino groups are acetylated, thepolymer is considered a polyacetylglucosamine. The degrees ofdeacetylation and functionalization impart a specific charge density tothe functionalized polyglucosamine derivative. The resulting chargedensity affects solubility and effectiveness of treatment. Thus, inaccordance with the present invention, the degree of deacetylation, thefunctionalization and the molecular weight must be optimized for optimalefficacy. The derivatized (i.e., functionalized) polyglucosaminesdescribed herein have a number of properties which are advantageousincluding solubility at physiologic pH.

A soluble polyglucosamine as described herein refers to a water solublechitosan or polyglucosamine that is not derivatized (i.e.,functionalized) on the hydroxyl or amine moieties other than with acetylgroups. A soluble polyglucosamine is comprised of glucosamine andacetylglucosamine monomers. Generally, a water soluble polyglucosaminehas a molecular weight of less than or equal to about 10,000 kDa (e.g.,less than or equal to about 5,000 kDa, e.g., less than or equal to about1,000 kDa) and a degree of deacetylation equal to or greater than 80%.In some embodiments, the molecular weight of the soluble polyglucosamineis between 5 and 1,000 kDa. In some embodiments, the molecular weight ofthe soluble polyglucosamine is between 10 and 350 kDa. In someembodiments, the molecular weight of the soluble polyglucosamine isbetween 15 and 200 kDa. In some embodiments, the molecular weight of thesoluble polyglucosamine is between 25 and 175 kDa. In some embodiments,the molecular weight of the soluble polyglucosamine is between 50 and150 kDa. The soluble polyglucosamines described herein are soluble at pH2 to pH 11.

The polyglucosamine derivatives described herein are generated byfunctionalizing the resulting free amino and or hydroxyl groups withpositively charged or neutral moieties, as described herein.

Polyglucosamines with any degree of deacetylation (DDA) greater than 50%are used in the present invention, with functionalization between 2% and50% of the available amines. The degree of deacetylation determines therelative content of free amino groups to total monomers in thepolyglucosamine polymer. Methods that can be used for determination ofthe degree of deacetylation of polyglucosamine include, e.g., ninhydrintest, linear potentiometric titration, near-infrared spectroscopy,nuclear magnetic resonance spectroscopy, hydrogen bromide titrimetry,infrared spectroscopy, and first derivative UV-spectrophotometry.Preferably, the degree of deacetylation of a soluble polyglucosamine ora derivatized (i.e., functionalized) polyglucosamine described herein isdetermined by quantitative infrared spectroscopy. Percentfunctionalization is determined as the % of derivatized (i.e.,functionalized) amines relative to the total number of available aminomoieties prior to reaction on the polyglucosamine polymer. Preferably,the percent functionalization of a derivatized (i.e., functionalized)polyglucosamine described herein is determined by H-NMR or quantitativeelemental analysis. The degrees of deacetylation and functionalizationimpart a specific charge density to the functionalized polyglucosaminederivative. The resulting charge density affects solubility, andstrength of interaction with cell membranes. The molecular weight isalso an important factor in the tenacity of cell membrane interaction.Thus, in accordance with the present invention, these properties must beoptimized for optimal efficacy. Exemplary polyglucosamine derivativesare described in U.S. Pat. No. 8,119,780, which is incorporated hereinby reference in its entirety.

The polyglucosamine derivatives described herein have a range ofpolydispersity index (PDI) between about 1.0 to about 2.5. As usedherein, the polydispersity index (PDI), is a measure of the distributionof molecular weights in a given polymer sample. The PDI calculated isthe weight averaged molecular weight divided by the number averagedmolecular weight. This calculation indicates the distribution ofindividual molecular weights in a batch of polymers. The PDI has a valuealways greater than 1, but as the polymer chains approach uniform chainlength, the PDI approaches unity (1). The PDI of a polymer derived froma natural source depends on the natural source (e.g. chitin or chitosanfrom crab vs. shrimp vs. fungi) and can be affected by a variety ofreaction, production, processing, handling, storage and purifyingconditions. Methods to determine the polydispersity include, e.g., gelpermeation chromatography (also known as size exclusion chromatography);light scattering measurements; and direct calculation from MALDI or fromelectrospray mass spectrometry. Preferably, the PDI of a solublepolyglucosamine or a derivatized (i.e., functionalized) polyglucosaminedescribed herein is determined by HPLC and multi angle light scatteringmethods.

The polyglucosamine derivatives (i.e., derivatized polyglucosamines orfunctionalized polyglucosamines) described herein have a variety ofselected molecular weights that are soluble at neutral and physiologicalpH, and include for the purposes of this invention molecular weightsranging from 5-1,000 kDa. Embodiments described herein are featuremedium range molecular weight of derivatized (i.e., functionalized)polyglucosamines (25 kDa, e.g., from about 15 to about 300 kDa). In someembodiments, the molecular weight of the derivatized (i.e.,functionalized) polyglucosamine is between 5 and 1,000 kDa. In someembodiments, the molecular weight of the derivatized (i.e.,functionalized) polyglucosamine is between 10 and 350 kDa. In someembodiments, the molecular weight of the derivatized (i.e.,functionalized) polyglucosamine is between 15 and 200 kDa. In someembodiments, the molecular weight of the functionalized polyglucosamineis between 25 and 175 kDa. In some embodiments, the molecular weight ofthe functionalized polyglucosamine is between 50 and 150 kDa (e.g.,between 50 and 125 kDa, e.g., between 60 to 100 kDa, e.g., about 90kDa).

The functionalized polyglucosamine derivatives described herein includethe following:

(A) Polyglucosamine-arginine compounds;

(B) Polyglucosamine-natural amino acid derivative compounds;

(C) Polyglucosamine-unnatural amino acid compounds;

(D) Polyglucosamine-acid amine compounds;

(E) Polyglucosamine-guanidine compounds; and

(F) Neutral polyglucosamine derivative compounds.

(A) Polyglucosamine-Arginine Compounds

In some embodiments, the present invention is directed topolyglucosamine-arginine compounds, where the arginine is bound througha peptide (amide) bond via its carbonyl to the primary amine on theglucosamines of polyglucosamine:

wherein each R¹ is independently selected from hydrogen, acetyl, and agroup of the following formula:

or a racemic mixture thereof, and

wherein at least 25% of R¹ substituents are H, at least 1% are acetyl,and at least 2% are a group of the formula shown above.

In some embodiments, a polyglucosamine-arginine compound is of thefollowing formula

where m is 0.02-0.50; q is 0.50-0.01; s is 1; p+q+m=1; the percentagedegree of functionalization is m·100%; and X is selected from the groupconsisting of:

wherein the preparation is substantially free of compounds having amolecular weight of less than 5000 Da.

(B) Polyglucosamine-Natural Amino Acid Derivative Compounds

In some embodiments, the present invention is directed topolyglucosamine-natural amino acid derivative compounds, wherein thenatural amino acid may be histidine or lysine. The amino is boundthrough a peptide (amide) bond via its carbonyl to the primary amine onthe glucosamines of polyglucosamine:

wherein each R¹ is independently selected from hydrogen, acetyl, and agroup of the following formula:

or a racemic mixture thereof, wherein at least 25% of R¹ substituentsare H, at least 1% are acetyl, and at least 2% are a group of theformula shown above; or a group of the following formula:

or a racemic mixture thereof, wherein at least 25% of R¹ substituentsare H, at least 1% are acetyl, and at least 2% are a group of theformula shown above.

(C) Polyglucosamine-Unnatural Amino Acid Compounds

In some embodiments, the present invention is directed topolyglucosamine-unnatural amino acid compounds, where the unnaturalamino acid is bound through a peptide (amide) bond via its carbonyl tothe primary amine on the glucosamines of polyglucosamine:

wherein each R¹ is independently selected from hydrogen, acetyl, and agroup of the following formula:

wherein R³ is an unnatural amino acid side chain, and wherein at least25% of R¹ substituents are H, at least 1% are acetyl, and at least 2%are a group of the formula shown above.

Unnatural amino acids are those with side chains not normally found inbiological systems, such as ornithine (2,5-diaminopentanoic acid). Anyunnatural amino acid may be used in accordance with the invention. Insome embodiments, the unnatural amino acid coupled to polyglucosamine isselected from one of the following:

(D) Polyglucosamine-Acid Amine Compounds

In some embodiments, the present invention is directed topolyglucosamine-acid amine compounds, or their guanidylatedcounterparts. The acid amine is bound through a peptide (amide) bond viaits carbonyl to the primary amine on the glucosamines ofpolyglucosamine:

wherein each R¹ is independently selected from hydrogen, acetyl, and agroup of the following formula:

wherein R³ is selected from amino, guanidino, and C₁-C₆ alkylsubstituted with an amino or a guanidino group, wherein at least 25% ofR¹ substituents are H, at least 1% are acetyl, and at least 2% are agroup of the formula shown above

In some embodiments, R¹ is selected from one of the following:

(E) Polyglucosamine-Guanidine Compounds

In some embodiments, the present invention is directed topolyglucosamine-guanidine compounds.

wherein each R¹ is independently selected from hydrogen, acetyl, and agroup in which R¹, together with the nitrogen to which it is attached,forms a guanidine moiety; wherein at least 25% of R¹ substituents are H,at least 1% are acetyl, and at least 2% form a guanidine moiety togetherwith the nitrogen to which it is attached.

(F) Neutral Polyglucosamine Derivative Compounds

In some embodiments, the present invention is directed to neutralpolyglucosamine derivative compounds. Exemplary neutral polyglucosaminederivative compounds include those where one or more amine nitrogens ofthe polyglucosamine have been covalently attached to a neutral moietysuch as a sugar:

wherein each R¹ is independently selected from hydrogen, acetyl, and asugar (e.g., a naturally occurring or modified sugar) or an α-hydroxyacid. Sugars can be monosaccharides, disaccharides or polysaccharidessuch as glucose, mannose, lactose, maltose, cellubiose, sucrose,amylose, glycogen, cellulose, gluconate, or pyruvate. Sugars can becovalently attached via a spacer or via the carboxylic acid, ketone oraldehyde group of the terminal sugar. Examples of α-hydroxy acidsinclude glycolic acid, lactic acid, and citric acid. In someembodiments, the neutral polyglucosamine derivative ispolyglucosamine-lactobionic acid compound or polyglucosamine-glycolicacid compound. Exemplary salts and coderivatives include those known inthe art, for example, those described in U.S. Pat. No. 8,119,780, thecontents of which is incorporated by reference in its entirety.

Formulations and Routes of Administration

The compounds described herein can be formulated in a variety ofmanners, including for topical delivery, oral delivery or delivery tothe GI tract. For example, the compounds can be administered, e.g.,topically (e.g., by solution (e.g., oral rinse, throat gargle, eyedrop), lotion, cream, ointment, gel, foam, transdermal patch, powder,solid, ponge, tape, vapor, inhalation or intranasal spray (e.g., nasalspray, nasal mists, sinus spray, nebulizer), enema, eye drops), orenterally (e.g., orally, gastric feeding tube, duodenal feeding tube,gastrostomy, rectally, buccally). Inclusion in feed, water or an inhaledformulation is particularly desirable for use with animals. In someembodiments, a compound is formulated so as to allow the solublepolyglucosamine or soluble polyglucosamine derivative thereof to diffuseinto a subject upon administration to the subject or to be ingested,inhaled or swabbed while incorporated into a time release formulation.

The compound described herein (e.g., a soluble polyglucosamine or aderivatized (i.e., functionalized) polyglucosamine) can be administeredbefore, during or after the onset of the condition or disorder describedherein. For example, the compound described herein can be administeredin a subject who has been treated or is being treated with radiationtherapy, e.g., other than that related to the treatment of cancer. Themethods herein contemplate administration of an effective amount ofcompound or compound composition to achieve the desired or statedeffect. The compounds can be administered as a continuous time-releaseor ad-libitim in water or food. Such administration can be used as anacute therapy (e.g., short-term treatment). The amount of activeingredient that may be combined with the carrier materials to produce asingle dosage form will vary depending upon the subject treated and theparticular mode of administration. A typical solution preparation willcontain from about 1 μg/mL to about 1000 μg/mL, about 5 μg/mL to about500 μg/mL, about 10 μg/mL to about 250 μg/mL, about 50 μg/mL to about200 μg/mL, or about 100 μg/mL to about 200 μg/mL of a compound describedherein, e.g., a compound of Formula (I). A typical solid diffusiblepreparation will contain from about 0.1% to about 10%, about 0.2% toabout 10%, or about 0.05% to about 5% by weight of a compound describedherein, e.g., a compound of Formula (I). A typical solid dissolvablepreparation will contain from about 0.1% to about 95%, about 0.2% toabout 70%, about 0.5% to about 40%, about 1% to about 10% by weight of acompound described herein, e.g., a compound of Formula (I).

Lower or higher doses than those recited above may be required. Specificdosage and treatment regimens for any particular patient will dependupon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the type andnature of the bacteria, the patient's disposition to the disease,condition or symptoms, and the judgment of the treating physician.

In some embodiments, the compounds described herein (e.g., a solublepolyglucosamine or a derivatized (i.e., functionalized) polyglucosamine)can be formulated, e.g., as a solution, gel, ointment, or dressing,e.g., for treating a subject that has been or will be exposed toradiation therapy, e.g., other than that related to the treatment ofcancer. In some embodiments, the dosage (e.g., solution dosage) is fromabout 10 μg/mL to about 1000 μg/mL, about 50 μg/mL to about 500 μg/mL,or about 100 μg/mL to about 300 μg/mL of a compound described herein,e.g., a compound of Formula (I), applied e.g., sufficiently to treat asubject that has been or will be exposed to radiation, trauma or shock.In some embodiments, the dosage (e.g., solution dosage) is from about 10to about 1000 μg/mL, about 50 μg/mL to about 500 μg/mL, or about 100μg/mL to about 300 μg/mL of a compound described herein, e.g., acompound of Formula (I), applied e.g., sufficiently to treat a subjectthat has been or will be exposed to radiation, trauma or shock at least1, 2, 3, 4, 5 or 6 times daily. In some embodiments, the soliddiffusible composition (dressing) is from about 0.1% to about 10%, about0.2% to about 8%, or about 0.5% to about 5%, by weight of a compounddescribed herein, e.g., a compound of Formula (I), applied e.g.,sufficiently to treat a subject that has been or will be exposed toradiation, trauma or shock at least 1, 2, 3, 4, 5 or 6 times daily.

In some embodiments, the compounds described herein (e.g., a solublepolyglucosamine or a derivatized (i.e., functionalized) polyglucosamine)can be formulated, e.g., as a solution, encapsulated time release, gel,or enema, e.g., for treating area a subject that has been or will beexposed to radiation, trauma or shock, e.g., in the mucous membrane,e.g., in the GI tract. In some embodiments, the dosage is from about 10μg/mL to about 1000 μg/mL, about 20 μg/mL to about 900 μs/mL, about 50μg/mL to about 500 μg/mL, about 60 μg/mL to about 300 μg/mL, or about 50to about 200 pg/mL of a compound described herein, e.g., a compound ofFormula (I), in solution, e.g., ad libitum, e.g., in water or fluid. Insome embodiments, the composition is administered at least 1, 2, 3, or 4times daily. In some embodiments, the dosage is from about 1 mg/kg toabout 200 mg/kg, about 2 mg/kg to about 100 mg/kg, about 4 mg/kg toabout 75 mg/kg, or about 5 mg/kg to about 40 mg/kg body weight of acompound described herein, e.g., a compound of Formula (I), in anencapsulated time release, gel, capsule or enema. In some embodiments,the composition is administered at least 1, 2, 3, 4, 5 or 6 times daily.

In some embodiments, the compounds described herein (e.g., a solublepolyglucosamine or a derivatized (i.e., functionalized) polyglucosamine)can be formulated as a nebulized solution or powder, or lavage, e.g.,for treating a subject that has been or will be exposed to radiation,trauma or shock, e.g., in respiratory tract. In some embodiments, thedosage is from about 500 μg to about 50000 μg, about 1000 μg to about25000 μg, about 2000 μg to about 10000 μg, or about 4000 μg to about6000 μg of a compound described herein, e.g., a compound of Formula (I),per kg body weight, every 2, 4, 6, 8, 10, 12, or 24 hours. In someembodiments, the composition is administered at least 1, 2, 3, 4, 5 or 6times daily.

In some embodiments, the compounds described herein (e.g., a solublepolyglucosamine or a derivatized (i.e., functionalized) polyglucosamine)can be formulated, e.g., as a spray, ointment, gel or inhalant, e.g.,for treating a disorder or condition described herein, e.g., in the GItract, throat, ear, or nose. In some embodiments, the dosage is fromabout 10 μg/mL to about 1000 μs/mL, about 20 μg/mL to about 500 μg/mL,about 50 μg/mL to about 300 μg/mL of a compound described herein, e.g.,a compound of Formula (I), in solution, about 0.1% to about 10%, about0.5% to about 5%, or about 1% to about 2%, by weight of a compounddescribed herein, e.g., a compound of Formula (I), in an ointment orgel. In some embodiments, the compound described herein, e.g., acompound of Formula (I), or composition is administered at least 1, 2,3, 4, 5 or 6 times daily.

In some embodiments, the compounds described herein (e.g., a solublepolyglucosamine or a derivatized (i.e., functionalized) polyglucosamine)can be formulated, e.g., as a solution, or encapsulated time release(e.g., enteric coating), e.g., for treating an inflammatorygastrointestinal disorder, e.g., as described herein. In someembodiments, the dosage is from about 0.1 to about 100 mg/kg bodyweight, about 1 to about 90 mg/kg body weight, about 10 to about 80mg/kg body weight, about 20 to about 70 mg/kg body weight, about 30 toabout 60 mg/kg body weight, about 0.1 to about 1 mg/kg body weight,about 1 to about 10 mg/kg body weight, about 10 to about 20 mg/kg bodyweight, about 20 to about 40 mg/kg body weight, about 40 to about 60mg/kg body weight, about 30 to about 50 mg/kg body weight (e.g., 40mg/kg body weight), about 60 to about 80 mg/kg body weight, or about 80to about 100 mg/kg body weight. In some embodiments, the composition isadministered at least 1, 2, 3, 4, 5 or 6 times daily.

Course of Treatment

Inventive methods of the present invention contemplate single as well asmultiple administrations of a therapeutically effective amount of acomposition as described herein. Compounds as described herein, e.g., acompound of Formula (I), can be administered at regular intervals. Insome embodiments, a composition described herein is administered in asingle dose. In some embodiments, a composition described herein isadministered in multiple doses.

In some embodiments, a therapeutically effective amount of a compound asdescribed herein, e.g., a compound of Formula (I), may be administeredperiodically at regular intervals (e.g., L 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore times every 1, 2, 3, 4, 5, or 6 days).

In some embodiments, a compositions described herein is administered ata predetermined interval (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moretimes every 1, 2, 3, 4, 5, or 6 days). In some embodiments, acomposition is administered once daily. In some embodiments, acomposition is administered from about 1 to about 5 times per day. Insome embodiments, a composition is administered from about 1 to about 3times per day.

In some embodiments, the subject is treated for up to 3 weeks (e.g., upto 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day).

In some embodiments, the subject is treated for 3 weeks, 2 weeks, 1week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day.

In some embodiments, the subject is treated within 1 hour, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours,20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or 2weeks after exposure to radiation, trauma, or shock.

It should also be understood that a specific dosage and treatmentregimen of any particular patient will depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health, diet, time of administration, rate of excretion,drug combination, and the judgment of the treating physician and theseverity of the disease or disorder treated. The amount of activeingredients will also depend upon the particular described compound andthe presence or absence and the nature of the additional agent in thecomposition.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained. Subjects may,however, require intermittent treatment on a long-term basis upon anyrecurrence of disease symptoms.

Pharmaceutical compositions of this invention comprise a compounddescribed herein, e.g., a compound of Formula (I), or a pharmaceuticallyacceptable salt thereof; an additional compound including for example, asteroid or an analgesic; and any pharmaceutically acceptable carrier,adjuvant or vehicle. Alternate compositions of this invention comprise acompound described herein, e.g., a compound of Formula (I), or apharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier, adjuvant or vehicle. The compositions delineatedherein include a compound described herein, e.g., a compound of Formula(I), as well as additional therapeutic compounds if present, in amountseffective for achieving a modulation of disease or disease symptoms.

The compositions are generally made by methods including the steps ofcombining a compound described herein with one or more carriers and,optionally, one or more additional therapeutic compounds delineatedherein.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, chewing gum, dissolving gel, emulsionsand aqueous suspensions, dispersions and solutions. In the case oftablets for oral use, carriers which are commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried corn starch. When aqueous suspensionsand/or emulsions are administered orally, the active ingredient may besuspended or dissolved in an oily phase which can be combined withemulsifying and/or suspending agents. If desired, certain sweeteningand/or flavoring and/or coloring agents may be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

In some cases, the pH of the formulation may be adjusted withpharmaceutically acceptable acids, bases or buffers to enhance thestability of the formulated compound or its delivery form for deliveryin particular regions of the body, such as the colon.

When the compositions of this invention comprise a combination ofcompounds described herein, both the compounds are generally present atdosage levels of between about 0.01 to 100%, and more preferably betweenabout 1 to 95% of the dosage normally administered in a monotherapyregimen. Additionally, combinations of a plurality of compoundsdescribed herein are also envisioned. The compounds may be administeredseparately, as part of a multiple dose regimen, from the compounds ofthis invention. The compounds may be administered in a manner and dosewhere they act synergistically, e.g., as described in US Publication No.2010-0130443, which is incorporated herein by reference in its entirety.Alternatively, those compounds may be part of a single dosage form,mixed together with the compounds of this invention in a singlecomposition.

Kits and Medical Devices

A compound described herein (e.g., a soluble polyglucosamine or aderivatized (i.e., functionalized) polyglucosamine) can be provided in akit. The kit includes (a) a composition that includes a compounddescribed herein, and, optionally (b) informational material. Theinformational material can be descriptive, instructional, marketing orother material that relates to the methods described herein and/or theuse of the compound described herein for the methods described herein.

The informational material of the kits is not limited in its form. Insome embodiments, the informational material can include informationabout production of the compound, molecular weight of the compound,concentration, date of expiration, batch or production site information,and so forth. In some embodiments, the informational material relates touse of the compound described herein to treat a disorder describedherein.

In some embodiments, the informational material can include instructionsto administer the compound described herein in a suitable manner toperform the methods described herein, e.g., in a suitable dose, dosageform, or mode of administration (e.g., a dose, dosage form, or mode ofadministration described herein). In another embodiment, theinformational material can include instructions to administer thecompound described herein to a suitable subject, e.g., a human, e.g., ahuman having or at risk for a disorder or condition described herein.For example, the material can include instructions to administer thecompound described herein to such a subject.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawing, and/or photograph,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as computer readable material,video recording, or audio recording. In another embodiment, theinformational material of the kit is contact information, e.g., aphysical address, email address, website, or telephone number, where auser of the kit can obtain substantive information about a compounddescribed herein and/or its use in the methods described herein. Ofcourse, the informational material can also be provided in anycombination of formats.

In addition to a compound described herein, the composition of the kitcan include other ingredients, such as a solvent or buffer, astabilizer, a preservative, and/or a second compound for treating acondition or disorder described herein. Alternatively, the otheringredients can be included in the kit, but in different compositions orcontainers than the compound described herein. In such embodiments, thekit can include instructions for admixing the compound described hereinand the other ingredients, or for using a compound described hereintogether with the other ingredients.

The compound described herein can be provided in any form, e.g., liquid,dried or lyophilized form. It may also be prepared as a capsule, pill,time-release or environment-release (e.g., pH sensitive) capsule orpill. It is preferred that the compound described herein besubstantially pure and/or sterile. When the compound described herein isprovided in a liquid solution, the liquid solution preferably is anaqueous solution, with a sterile aqueous solution being preferred. Whenthe compound described herein is provided as a dried form,reconstitution generally is by the addition of a suitable solvent. Thesolvent, e.g., sterile water or buffer, can optionally be provided inthe kit.

The kit can include one or more containers for the compositioncontaining the compound described herein. In some embodiments, the kitcontains separate containers, dividers or compartments for thecomposition and informational material. For example, the composition canbe contained in a bottle, vial, or syringe, and the informationalmaterial can be contained in a plastic sleeve or packet. In otherembodiments, the separate elements of the kit are contained within asingle, undivided container. For example, the composition is containedin a bottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some embodiments, the kit includes aplurality (e.g., a pack) of individual containers, each containing oneor more unit dosage forms (e.g., a dosage form described herein) of acompound described herein. For example, the kit includes a plurality ofsyringes, ampules, foil packets, or blister packs, each containing asingle unit dose of a compound described herein. The containers of thekits can be air tight, waterproof (e.g., impermeable to changes inmoisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for administration of thecomposition, e.g., a syringe, dosing bottle, single-unit dosingpreparation, pipette, measured spoon, dropper (e.g., eye dropper), swab(e.g., a cotton swab or wooden swab), or any such delivery device.

The composition described herein can be used in a medical device fortreating a subject e.g., a mucosal surface of a subject, that has beenor will be exposed to radiation, trauma or shock.

EXAMPLES

As provided in the Examples below, PAAG refers to poly (acetyl, arginyl)glucosamine with an average molecular weight of 86 kDa and 30%functionalized unless indicated otherwise. A fraction of the amines ofthe glucosamine on the polyglucosamine are reacted with a singlearginine, as opposed to a dimer, trimer or larger polyarginine. Thismonoacetylation of each reacted amine is accomplished by using aprotecting group on the primary amine of the arginine upon coupling asdescribed in U.S. Pat. No. 8,119,780, the contents of which areincorporated herein by reference.

As shown in the Examples below, in vivo data (e.g., increased survival)suggest improved recovery and reduced morbidity. Furthermore, in vitrodata (e.g., down-regulated IL-8) suggest the generation of a lesspro-inflammatory environment.

These exemplary results demonstrate that PAAG has the ability to treatand prevent damage resulting from radiation, trauma or shock. Further,it enhances the healing rate of areas that have been exposed toradiation, trauma or shock.

Example 1: Total Body Irradiation Study Methods

Male mice were exposed to an acute irradiation dose on Day 0 of 0, 12.5,or 13.0 Gy at a rate of 1 Gy/min with lead shielding to the left hindlimb. Starting 24 hours after irradiation, the mice were untreated ortreated with PAAG or vehicle control, given with 50 mg/kg via oralgavage daily and dosed ad libitum in the drinking water (200 ppm PAAG)from days 1 to 18. The treatment groups are summarized on TABLE 1.

TABLE 1 Dosing (in Measure Male Radiation drinking Survival Mice Dose onDosing water) and Body Group (n) Day 0 Treatment (p.o.) Days 1-18 Weight1 5 None None None None Days 0-18 2 5 None PAAG q.d. PAAG Days 0-18 50mg/kg Days 1-18* 200 ppm 3 16 12.5 Gy Vehicle Control q.d. None Days0-18 Days 1-18* 4 16 12.5 Gy PAAG q.d. PAAG Days 0-18 50 mg/kg Days1-18* 200 ppm 5 16 13.0 Gy Vehicle Control q.d. None Days 0-18 Days1-18* 6 16 13.0 Gy PAAG q.d. PAAG Days 0-18 50 mg/kg Days 1-18* 200 ppm*The first dose was administered 24 hours after irradiation.PAAG Treatment Reduces Mortality in Mice Dosed with Radiation

The total body irradiation study was performed as described above withcontrol vehicle or PAAG (86 kDa and 30% functionalization) administeredto the mice at the indicated final concentrations. The results of thestudy showed a dramatic reduction in mortality for mice exposed to an LD80 dose of ionizing radiation. As shown in the Kaplan-Meier SurvivalPlot in FIG. 1, there was no mortality in animals that were notirradiated. No statistical difference was observed between the treatedand untreated groups exposed to 12.5Gy of radiation (p=0.081), an LD 20.However, a very significant (p<0.0001) difference was observed in the LD80 dose in mice given vehicle as compared to PAAG. In fact, 88% of thetreated animals survived while only 19% of the untreated animalssurvived. As shown in FIG. 2, the corresponding plot of the mean percentweight change shows PAAG has no significant effect on weight change inanimals as compared with untreated animals. The comparative mortalitydata from these experiments is tabulated on TABLE 2.

TABLE 2 Percent Mice per No. of Day of Observation/ Dead Per GroupRadiation Treatment Group (n) Death Death Action^(†) Group 1 None NoneNone 0 — — 0 2 None Chitosan-  5 0 — — 0 Arginine 3 12.5 Gy Vehicle 16 1Day 6 Euthanized 6.25 Control 4 12.5 Gy Chitosan- 16 1 Day 5 Euthanized31.25 Arginine 1 Day 6 Euthanized 3 Day 7 Euthanized 5 13.0 Gy Vehicle16 2 Day 5 Euthanized 81.25 Control 5 Day 6 Euthanized 5 Day 6 Founddead 1 Day 7 Euthanized 6 13.0 Gy Chitosan-  15* 1 Day 6 Found dead13.33^(¶) Arginine 1  Day 18 Euthanized *Group 6 had 1 animal excludedfrom analysis as a result of non-treatment related euthanasia due tooral gavage injury. ^(†)Euthanasia due to moribund or body weightloss >30% from baseline. ^(¶)Based on n = 15.

Example 2: Mucosal Radiation Dose Ranging Study Methods

In a dose ranging study, an acute dose of irradiation of 40 Gy directedto the left buccal cheek pouch of Syrian Golden hamsters and thehamsters were treated t.i.d. with 50, 100, 250 and 500 μg/mL PAAG orvehicle control directly inserted into the pouch. Mean % weight changeand mucositis score were recorded.

PAAG Modulated the Course of Oral Mucositis

The mucosal radiation dose ranging study was performed as describedabove with PAAG (86 kDa and 30% functionalization) added to theindicated final concentrations. Comparison of the mean mucositis scoresfor a vehicle control and four doses of PAAG are shown in FIG. 3.Consistent with its attenuation of chemically-induced intestinal injury(data not shown), PAAG modulated the course of oral mucositis. Acomparison of the mean percent weight gain for mice dosed with vehiclecontrol and four dosing concentrations of PAAG is shown in FIG. 4.Percent ulceration as shown by a plot of the percent of animals with amucositis score of 3 or higher on a given day post irradiation for avehicle control and two doses of PAAG is shown in FIG. 5. The healingeffect of PAAG is shown in FIG. 6, which depicts the comparison of thepercentage of animals with a mucositis score of 3 or higher on a givenday post irradiation for a vehicle control and three doses of PAAG.

Example 3: Oral Mucositis Scheduling Study Methods

In a scheduling study, an acute dose of irradiation of 40 Gy directed tothe left buccal cheek pouch and the hamsters were treated t.i.d. with200 μg/mL PAAG or vehicle control directly inserted into the pouch.Schedules included PAAG treatment Day −7 to 36, Day −1 to 36, Day −1 to14, and Day 10-36. Mean % weight change and mucositis score wererecorded.

Optimization of Scheduling

Oral mucositis scheduling study was performed as described above withPAAG (86 kDa and 30% functionalization) added to the indicated finalconcentrations. The mean mucositis scores for a vehicle control and 200ppm PAAG administered thrice daily from Day −7 to 36, Day −1 to 36, Day−1 to 14, or Day 10 to 36 is shown in FIG. 7. A comparison of the meanpercent weight gain for mice dosed with vehicle control and 200 ppm PAAGfrom Day −7 to 36, Day −1 to 36, Day −1 to 14, or Day 10 to 36 is shownin FIG. 8. Mice dosed with 200 ppm PAAG administered thrice daily fromDay −1 to 14 or from Day 10 to 36 have a reduced mean mucositis scorerelative to mice dosed with 200 ppm PAAG thrice daily from Day −7 to 36or from Day −1 to 36. Shown in FIG. 9 is a comparison of the percentageof animals with a score of 3 or greater in mice dosed with a vehiclecontrol and those dosed with 200 ppm PAAG administered thrice daily atDay −1 to 14 and Day 10 to 36.

Example 4: IL-8 Production Response Study Methods

The human myeloid cell line (U937) was propagated in RPMI 1640supplemented with 10% (v/v) fetal bovine serum and 2 mm L-glutamine.Cells were seeded at 6×10⁵ cells per well in 24 well plates in RPMI 1640additionally supplemented with 0.1 ug PMA for 48 hours to activate U937cells to be macrophage-like. Cell media was replaced with media withoutfetal bovine serum and PMA for at least 2 hours. The duration ofpretreatments of Lactoferrin (100 ng/ml), and PAAG (200 ug/ml) was onehour before cells and media being rinsed twice with D-PBS. Cells werethen subjected to media containing LPS (10 ng/ml) for IL-8 stimulation.Supernatants were extracted and stored after 4 hours from the time ofLPS stimulation. An IL-8 ELISA was performed according to the BioLegendprotocol to measure IL-8 production.

Production of I-L8 in U937 Cells Following Stimulation with LPS isReduced when Pretreated with 100 ppm PAAG

IL-8 production was performed as described, with PAAG (86 kDa and 30%functionalization), LPS, and Lactoferrin added to the indicated finalconcentrations prior to rinsing and activation by LPS. As shown in FIG.10, pretreatment with PAAG reduced the LPS activated IL-8 responserelative to the response pretreated with control or lactoferrin.

Example 5: Nasal Epithelium/MRSA Binding Assays Methods

The nasal epithelium cell line (RPMI-2650) were grown in Eagle's MinimalEssential Media (EMEM) supplemented with 10% (v/v) fetal bovine serumand 2 mm L-glutamine. RPMI 2650 cells were seeded at 2.5×10⁵ per well in24 well plates and incubated at 37° C. and in atmosphere of 5% CO₂ for24 hours. The cells were then rinsed twice with D-PBS and replaced with(EMEM) without fetal bovine serum for another 24 hours. An overnightculture of MRSA strain MW2 (ATCC BAA-1707) using LB-broth was grown 24hours before inoculation. PAAG (86 kDa and 30% functionalization) wasdissolved in either EMEM without fetal bovine serum or D-PBS in theirrespective concentrations for pretreatment. Pretreatment of PAAGconsisted of rinsing cells of prior cell culture media with D-PBS onceand adding media with 200 or 500 ppm PAAG for either 5 min or one hourand with 200 or 500 ppm PAAG. Upon completion of pretreatment, cellswere rinsed twice with D-PBS to remove any non-adherent PAAG andinoculated with MW2 in growth phase at an MOI of 1:100 for one hour.Cells were then rinsed twice with D-PBS before being lysed using 0.5%Triton X-100 in D-PBS. Supernatants were extracted and serially diluted,plated and counted for bacterial attachment.

Pretreatment of PAAG Decreases Binding of MRSA to Nasal Epithelial Cells

Pretreatment and binding studies were performed as described above, withthe PAAG added to the indicated final concentrations. As shown on FIG.11, pretreatment of cells with 200 or 500 ppm PAAG for 5 and 60 minutesdecreases binding of MRSA to nasal epithelial cells as compared to PBSand media controls.

Example 6: Bacterial Attachment or Invasion Study Methods

Caco2 Attachment.

The gut epithelium cell line (Caco2) were grown in Eagle's MinimalEssential Media (EMEM) supplemented with 10% (v/v) fetal bovine serumand 2 mm L-glutamine. Caco2 cells were seeded at 2.5×10⁵ per well in 24well plates and incubated at 37° C. and in atmosphere of 5% CO₂ for 24hours to confluency. The cells were then rinsed twice with D-PBS andreplaced with (EMEM) without fetal bovine serum for another 24 hours.Cells were rinsed with D-PBS twice and replaced with media containing nofetal bovine serum. Pretreatment of the cells with PAAG 200 ug/ml (86kDa, 30% functionalization; 37 kDa. 22% functionalization; and 27 kDa,21% functionalization) dissolved in media without serum for one hour wasperformed and the cells were then rinsed twice with D-PBS to remove anynon-adherent PAAG. After rinsing, the cells were inoculated withAcinetobacter baumaunii in growth phase at a multiplicity of infection(MOI) of 1:100 for one hour. Cells were then rinsed twice with D-PBSbefore being lysed using 0.5% Triton X-100 in D-PBS. Supernatants wereextracted and serially diluted, plated and counted for bacterialattachment.

Burkholderia cepacia Macrophage Uptake.

The myeloid cell line (U937) were grown in RPMI 1640 supplemented with10% (v/v) fetal bovine serum and 2 mm L-glutamine. Cells were seeded at6×10⁵ cells per well in 24 well plates in RPMI 1640 additionallysupplemented with 0.1 ug PMA for 48 hours to activate U937 cells to bemacrophage-like. Cell media was replaced with cell media without fetalbovine serum and PMA for at least 2 hours. Treatments consisted ofeither PAAG (200 ug/ml) treated on cells for one hour and being rinsedwith D-PBS twice before inoculation or with PAAG treatment with norinse. Upon completion of pretreatment with PAAG, cells were inoculatedwith Burkholderia cepacia in growth phase with an MOI of 1:10 for 45min. After inoculation period media is supplemented with 50 ug/ml ofgentamicin for another 45 min. Cells were then rinsed twice with D-PBSbefore being lysed using 0.5% Triton X-100 in D-PBS and seriallydiluted, plated and counted for bacterial uptake.

Results

Pretreatment and binding studies were performed as described above forCaco2 cells with Acinetobacter attachment and Burkholderia cepaciamacrophage uptake studies, with the PAAG (86 kDa and 30%functionalization) added to the indicated final concentrations. As shownin FIG. 12A, pretreatment of Caco2 cells with 200 μg/mL PAAG decreasesthe percentage of bacteria bound to Caco2 cells relative to untreatedcells, as measured by inoculum present in extracted supernatant.

As shown in FIG. 12B, pretreatment of the myeloid cell line (U937) with200 μg/mL PAAG decreases the percentage of bacterial invasion byBurkholderia cepacia relative to untreated cells.

Example 7: Examination of Protective Effect of PAAG on DamagedEpithelial Cells Methods

A431 epidermal cells were seeded into 4-well chamber slides in DMEM plus10% FBS at a density to be confluent the next day (5×10⁵/well (˜1.8cm²)). The following day, a scratch was made across the confluentmonolayer using a sterile 10 μl tip. The medium was aspirated and thewells rinsed with DMEM to remove floating debris. Serum free DMEM wasadded to all wells, plus or minus PAAG (18 kD, 25% functionalization) orEGF as a positive control. Cells were treated for 24, 48, 72 or 96hours. Cells were fixed with paraformaldehyde at the indicated times andstained with hematoxylin for better visualization. A representativescratch at time 0 is duplicated at the top of each column of treatmentsfor comparison depicted in FIG. 13.

Results

Scratches “heal” faster than control with either the PAAG or EGF. Thecombination of PAAG and EGF together resulted in even faster healing,suggesting that PAAG works with EGF to enhance cellular EGF activity.This observation has been reproduced in a number of different cell linesand in in vivo animal models of mucosal damage. Interestingly, thiseffect is not observed in subconfluent or non-damaged monolayers.

Example 8: Necrotic Enteritis Model Poultry Study of Mortality Methods

In a lethal necrotic enteritis model poultry study of over 1000 birds,PAAG was given orally in the drinking water to chicks infected withClostridium perfringens (CP) after sensitization by coccidia. Doses ofPAAG (22 kD, 36% functionalization) in water ad libitum were given 1 dayprior to CP infection and for 5 days post infection. Mortality wasassessed 14 days after infection.

Results

While 32% of the control animals died, only 15% of the treated animalsdied as shown in FIG. 14. Bacitracin and 100 ppm PAAG delivered adlibitum in water reduced morality with statistical significance relativeto control. 10 ppm was numerically insignificant versus control.

Example 9: Evaluation of PAAG-Mediated Radioprotection on IntestinalBacterial Translocation in C57BL/6 Mice Study Design

Seventy two C57BL/6 mice were enrolled in this experiment. Mice wererandomized into four groups of eighteen animals each: non-irradiatedcontrols and active PAAG, irradiated control and active PAAG. On day 0,mice in groups 3 and 4 were exposed to 13.0 Gy radiation at a rate of1.0 Gy/min (with 5% long bone protection). Mice were treated accordingto TABLE 3. PAAG (50 mg/kg) or vehicle was administered in drinkingwater. Six animals were euthanized at 2, 3, and 4 days after radiationexposure, and the ileum and jejunum were fixed then analyzed by apathologist blinded to the sample identification. At the time ofeuthanasia, their small intestines were removed, flushed with saline,and divided into three segments. The most proximal 5 cm from the firstsegment, middle 7 cm from the middle segment, and distal 5 cm from thethird segment were removed, flushed with saline, and the middle 3 cmfrom each excised and snap frozen. Remaining tissue was placed in 10%neutral buffered formalin for histology. The mesenteric lymph nodes(MLN) were also collected from all the animals and assessed as the full6 animal sample set for total bacterial count at day 4.

TABLE 3 Radiation Euthanasia N at Each Group Dose Treatment Route Timepoints Time Point 1 n/a n/a n/a Days 2, 3, 4 6 2 n/a PAAG PO Days 2, 3,4 6 50 mg/kg 3 13.0 Gy Vehicle PO Days 2, 3, 4 6 4 13.0 Gy PAAG PO Days2, 3, 4 6 50 mg/kg

Tissue Examination

Small intestinal segments were removed at necropsy and fixed asdescribed above. Tissues were embedded in paraffin, sectioned at 5microns, and stained with hematoxylin and eosin (HE) and gram stain.Slides were evaluated for 4 parameters by a board certified veterinarypathologist as below.

The % of epithelial loss was estimated to the nearest 10%. Inflammation,crypt loss, and crypt regeneration were scored on a scale of 0-4 where0=no change, 1=mild change, 2=moderate change, 3=marked change, and4=severe change.

Results

FIGS. 15-27 depict the data generated for the study. In all graphs barsrepresent group means with standard errors shown. Statistical analysiswas performed using GraphPad Prism software and statistical significanceis noted in the chart and/or tables when appropriate.

PAAG treatment partially protected C57BL/6 mice from thegastrointestinal effects of total body irradiation. Irradiated,vehicle-treated mice had marked crypt degeneration evident from 2-4 dayspost-irradiation. Crypt degeneration was associated with inflammationprogressing from acute to chronic over the course of the experiment.Without crypt epithelial cells to replenish villous epithelium, villibecame progressively shorter and were lined by more degenerate cells atlater time points. In contrast, PAAG-treated mice had less severe cryptdegeneration, reduced inflammation, and tended to retain healthiervillous epithelium. The results of bacterial counts for the MLNdemonstrate a dramatic reduction of circulating bacteria in the case ofthe treated mice. In FIG. 16, the colony forming units (CFU)/g ofbacteria in the MLN for all 6 animals in the irradiated andnon-irradiated groups is shown. For the irradiated animals, the PAAGtreated group had dramatically less bacteria in the MLN than in thevehicle control group.

Quantitative analysis of the histological data at day 4 shows that PAAGreduced GI inflammation, reduced epithelial loss and reduced crypt lossrelative to control in mice exposed to lethal ionizing radiation (FIG.19). These findings support a radio-protective effect of PAAG whichprevious reports have linked to free radical scavenging (Nishimura etal, 2003) or to mucosal-protective properties (Ersin et al, 2000).

Data from day 4, in both groups and more severe in vehicle-treatedanimals, suggest susceptibility to bacterial translocation acrossdamaged intestinal mucosa coincident with a loss of villous epithelium.Prior to day 4, crypt damage may be insufficient to cause significantbacterial infection and translocation to deeper tissue layers.

Description and Representative Photomicrographs:

Group 1, No Treatment+No Radiation:

All regions of small intestine were essentially normal.

Group 2, PAAG+No Radiation:

All regions of small intestine were essentially normal.

Group 3, Vehicle-Treatment+13 Gy Radiation:

All regions of the small intestine were affected with similar findings.

At day 2, there was moderate to marked crypt degeneration. Crypt liningcells were apoptotic after radiation injury. Nuclei were pyknotic,cytoplasm was shrunken and condensed, and cells detached from theunderlying basement membrane and sloughed into the crypt lumen. Somecells had characteristics more suggestive of necrosis with lysis of cellwalls. This tended to be in areas of active inflammation suggesting thatinflammatory mediators may have played a role in cell necrosis.Inflammation was mixed with large numbers of neutrophils admixed withmacrophages and lymphocytes. Overall, villi retained their normal heightand structure and their epithelium was intact.

At day 3, there was progressive crypt degeneration and loss. In somecrypts, epithelial cells were apoptotic or necrotic as described above.In other areas, there was simply a loss of crypts and replacement bylymphohistiocytic inflammation. There was mild crypt regenerationcharacterized by large, intensely basophilic cells with large nuclei andactive mitoses. Villi were slightly blunted and villous epithelium wasmildly degenerate.

At day 4, there was more severe loss of villous epithelium. The markedcrypt loss was as would be expected for animals with chronicinflammation and early fibrosis. As opposed to the neurophils seen atday 2, inflammation was predominantly characterized by histiocytesadmixed with lymphocytes. Some crypts were still degenerating (thickarrow in jejunum in FIG. 25), but overall there was simply a loss ofnormal crypts along the base of villi. Villi were shortened and bluntedand the epithelium was vacuolated and degenerate. There was, however,regeneration of some crypts which was especially evident in the jejunum.Regenerating crypts (arrows in FIG. 25) are deeply basophilic, havelarge cells, and mitoses are often visible. In general, crypts wereabsent rather than overtly degenerate and the base of villi was lined bya mixed inflammatory cell population and fibroblasts instead of crypts.Crypt regeneration continued multifocally but was mild overall.

Group 4, PAAG-Treatment+13 Gy Radiation:

All regions of the small intestine were affected with similar findings.

At day 2, there was mild to moderate crypt degeneration. Crypt liningcells were apoptotic after radiation injury. Nuclei were pyknotic,cytoplasm was shrunken and condensed, and cells detached from theunderlying basement membrane and sloughed into the crypt lumen. Somecells had characteristics more suggestive of necrosis with lysis of cellwalls. This tended to be in areas of active inflammation suggesting thatinflammatory mediators may have played a role in cell necrosis.Inflammation was mixed with scattered foci of neutrophils admixed withmacrophages and lymphocytes. Overall, villi retained their normal heightand structure and their epithelium was intact.

At day 3, there was progressive crypt degeneration and loss. In somecrypts, epithelial cells were apoptotic or necrotic as described above.In other areas, there was simply a loss of crypts and replacement bylymphohistiocytic inflammation. There was mild to moderate cryptregeneration characterized by large, intensely basophilic cells withlarge nuclei and active mitoses. Villi were minimally blunted.

As in Group 3 animals, there was an increased loss of villous epitheliumat day 4, but this loss was not as severe as in group 3 animals. Cryptloss was moderate to marked and associated with chronic inflammation andearly fibrosis. As opposed to the neutrophils seen at day 2,inflammation was predominantly characterized by histiocytes admixed withlymphocytes. There was moderate to marked crypt regeneration, which wasmore prominent in the jejunum, and in some cases regenerating cellscould be seen spreading onto the base of villi. For example,regenerating crypts (arrows in FIG. 27) are deeply basophilic, havelarge cells, and mitoses are often visible. Villi were shortened andblunted as in Group 3, but the epithelium is less degenerate.Inflammation was more chronic in nature and replaced cryptsmultifocally.

See FIGS. 22-27 for representative photomicrographs.

Summary

PAAG treatment partially protected C57BL/6 mice from the GI effects oftotal body irradiation. Irradiated, vehicle-treated mice had markedcrypt degeneration evident from 2-4 days post-irradiation. Cryptdegeneration was associated with inflammation progressing from acute tochronic over the course of the experiments. Villi became progressivelyshorter and were lined by more denegerate cells at later time points,which would be expected without crypt epithelial cells to replenishvillous epithelium. In contrast, PAAG-treated mice had less severe cryptdegeneration, reduced inflammation, and tended to retain healthiervillous epithelium, suggesting a radio-protective effect of PAAG.

Example 10: Evaluation of Effect of PAAG on Irradiation BiomarkersMethods

Control or irradiated mice were treated with vehicle or PAAG once a dayvia oral gavage at day 2, 3, and 4 starting 24 after total bodyirradiation with 5% long bone protection.

Pro-Calcitonin Levels in Plasma are not Affected by PAAG

Pro-calcitonin (PCT) is a circulating plasma marker that has been usedto indicate systemic inflammation and has been used in radiation studiesto detect GI inflammation. PCT serum levels have been shown to increasein relation to the magnitude of bacteremia in mice.

PAAG reduced circulating plasma levels of PCT relative to controls inmice exposed to a potentially lethal dose of ionizing radiation (13 Gy),as shown on FIG. 28.

Citrulline Levels are Increased in Plasma by Treatment with PAAG

Citrulline is an unnatural amino acid and metabolic end product producedby small bowel enterocytes (cells specific to the GI tract). Levels ofcitrulline are reduced when enterocytes are damaged or reduced.Citrulline is mainly produced from viable enterocytes of the small boweland has been used for quantifying radiation-induced epithelial cell loss(Lutgens 2003, Int. J. Rad. Oncol. Biol. Phys. 57 (4), 1067) and as amarker of GI damage. Decreased serum citrulline levels correlate withloss of cell viability or damage to the bowel.

PAAG increased circulating amounts of citrulline relative to control onday 3 and 4 in mice relative to control after exposure to lethalionizing radiation, as shown on FIG. 29. Plasma citrulline wasdramatically reduced upon animal exposure to 13 Gy radiation, however,PAAG treated irradiated animals had statistically less citrullinereduction than untreated irradiated animals (P values below), indicatingless epithelial cell loss. This result indicates that administration ofPAAG reduces damage to enterocytes in the GI tract.

Example 11: Kaplan-Mayer Survival Plot after Three Doses of RadiationTreated with Vehicle or PAAG

Male CB57BL/6 mice of the same weight and age were subjected toradiation doses of 12.5, 13.5 and 14.0 Gy. Animals were given the samedose of PAAG (50 mg/kg once a day) by oral gavage as in the previousstudy. Eighteen (18) animals were studied in each of 8 arms: irradiatedcontrol and active PAAG at each dose. Mortality data is shown in FIG. 30for three doses of total body radiation treated with vehicle or PAAGgiven via oral gavage daily. 12.5 Gy dose treated with vehicle or PAAGare numerically significant. 13.5 Gy dose treated with vehicle or PAAG,or 14.0 Gy dosed with vehicle or PAAG are not statistically different.

This study confirms the study shown above in Example 1 suggesting thatthe activity of PAAG reduced mortality after significant LD70 ionizingradiation. The study also suggests that when the damage is too great,PAAG may not be able to overcome the overwhelming inflammatory responseof the GI tract. No adverse effects were observed at any dose.

1. A method of treating a subject, the method comprising identifying asubject that has been exposed to radiation, trauma or shock, andtreating the subject by administering a therapeutically effective amountof a compound to the subject, wherein the compound is a compound ofFormula (I):

wherein: n is an integer between 20 and 6000; and each R¹ isindependently selected for each occurrence from hydrogen, acetyl,

wherein at least 25% of R¹ substituents are H, at least 1% of R¹substituents are acetyl, and at least 2% of R¹ substituents are

wherein upon administration of the compound, the compound treats,reduces the severity or delays the onset of sepsis or reduces thelikelihood of mortality in the subject, thereby treating the subject. 2.The method of claim 1, wherein the subject has a bacterial infection,chemical damage or radiation damage.
 3. The method of claim 2, whereinthe infection or damage results in leaky gut.
 4. The method of claim 1,wherein the method reduces the severity of sepsis or a symptom thereofor decreases the likelihood of mortality from the radiation, trauma orshock relative to a subject not administered with the compound.
 5. Themethod of claim 1, wherein the subject has sepsis or a symptom of sepsisresulting from radiation, trauma or shock. 6-8. (canceled)
 9. The methodof claim 1, wherein the trauma or shock is a bacterial, viral, or fungalinfection resulting in GI damage.
 10. The method of claim 9, wherein thebacterial infection is from one of the following bacteria: Salmonellaenterica serovar Typhimurium, Shigella flexneri, E. coli and P.aeruginosa.
 11. The method of claim 1, wherein the method reducesinflammation in the subject from the radiation, trauma or shock. 12-17.(canceled)
 18. The method of claim 1, wherein the trauma results fromexposure to a toxic chemical or poison.
 19. (canceled)
 20. The method ofclaim 1, wherein the trauma results from multi-organ failure fromphysical damage and trauma to the body, burns, blast injury, systemicinfection, blood loss (hypotension), and traumatic brain injury.
 21. Themethod of claim 1, wherein the shock results from excessive stimuli,commensals, injury, heat, autoantigens, tumors, or necrotic cells.22-25. (canceled)
 26. The method of claim 1, wherein the subject hasnecrotizing entercolitis, necrotic enteritis, short bowel syndrome orshort gut syndrome. 27-29. (canceled)
 30. The method of claim 1, whereinthe compound is administered at regular intervals. 31-33. (canceled) 34.The method of claim 1, wherein the subject is treated within 1 hour, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours,16 hours, 20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1week, or 2 weeks after exposure to radiation, trauma, shock, orinfection.
 35. The method of claim 1, wherein the derivatized chitosanis functionalized at between 18% and 30%.
 36. The method of claim 1,wherein the molecular weight of the derivatized chitosan is from 50 to150 kDa.
 37. The method of claim 1, wherein the polydispersity index ofthe derivatized chitosan is from 1.0 to 2.5.
 38. (canceled)
 39. A methodof reducing permeability of the gastrointestinal tract of a subject, themethod comprising identifying a subject that has been exposed toradiation, trauma or shock, and treating the subject by administering atherapeutically effective amount of a compound to the subject, whereinthe compound is a compound of Formula (I):

wherein: n is an integer between 20 and 6000; and each R¹ isindependently selected for each occurrence from hydrogen, acetyl,

wherein at least 25% of R¹ substituents are H, at least 1% of R¹substituents are acetyl, and at least 2% of R¹ substituents are

wherein upon administration of the compound, the compound reducespermeability of the gastrointestinal tract of a subject, therebytreating the subject.
 40. The method of 39, wherein the permeability isa result of shock, trauma, or exposure to infection in the GI.